Process of Preparing Guanylate Cyclase C Agonists

ABSTRACT

The invention provides processes of preparing a peptide including a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249 described herein.

RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.application Ser. No. 15/405,787 filed Jan. 13, 2017, which is acontinuation of, and claims priority to, U.S. application Ser. No.14/001,638 filed Feb. 11, 2014, which is a 35 U.S.C. § 371 NationalPhase Application of and claims priority to PCT/US2012/027287, filedMar. 1, 2012 which claims priority to U.S. Provisional Application No.61/447,891 filed Mar. 1, 2011, the contents of each of which areincorporated by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “376464-2003US3 SequenceListing.txt”, which was created on Aug. 21, 2019 and is 112 KB in size,are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to processes of preparing guanylatecyclase C peptide agonists useful for the treatment and prevention ofvarious diseases and disorders.

BACKGROUND OF THE INVENTION

Guanylate cyclase C is a transmembrane form of guanylate cyclase that isexpressed on various cells, including gastrointestinal epithelial cells(reviewed in Vaandrager 2002 Mol. Cell. Biochem. 230:73-83). It wasoriginally discovered as the intestinal receptor for the heat-stabletoxin (ST) peptides secreted by enteric bacteria and which causediarrhea. The ST peptides share a similar primary amino acid structurewith two peptides isolated from intestinal mucosa and urine, guanylinand uroguanylin (Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951(1992); Hamra, et al., Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993);Forte, L., Reg. Pept. 81:25-39 (1999); Schulz, et al., Cell 63:941-948(1990); Guba, et al., Gastroenterology 111:1558-1568 (1996); Joo, etal., Am. J. Physiol. 274:G633-G644 (1998)).

In the intestines, guanylin and uroguanylin act as regulators of fluidand electrolyte balance. In response to high oral salt intake, thesepeptides are released into the intestinal lumen where they bind toguanylate cyclase C localized on the luminal membrane of enterocytes(simple columnar epithelial cells of the small intestines and colon).The binding of the guanylin peptides to guanylate cyclase C induceselectrolyte and water excretion into the intestinal lumen via a complexintracellular signaling cascade that is initiated by an increase incyclic guanosine monophosphate (cGMP).

The cGMP-mediated signaling that is initiated by the guanylin peptidesis critical for the normal functioning of the gut. Any abnormality inthis process could lead to gastrointestinal disorders such as irritablebowel syndrome (IBS) and inflammatory bowel diseases. Inflammatory boweldisease is a general name given to a group of disorders that cause theintestines to become inflamed, characterized by red and swollen tissue.Examples include ulcerative colitis and Crohn's disease. Crohn's diseaseis a serious inflammatory disease that predominantly affects the ileumand colon, but can also occur in other sections of the gastrointestinaltract. Ulcerative colitis is exclusively an inflammatory disease of thecolon, the large intestine. Unlike Crohn's disease, in which all layersof the intestine are involved, and in which there can be normal healthybowel in between patches of diseased bowel, ulcerative colitis affectsonly the innermost lining (mucosa) of the colon in a continuous manner.Depending on which portion of the gastrointestinal tract is involved,Crohn's disease may be referred to as ileitis, regional enteritis,colitis, etc. Crohn's disease and ulcerative colitis differ from spasticcolon or irritable bowel syndrome, which are motility disorders of thegastrointestinal tract. Gastrointestinal inflammation can be a chroniccondition. It is estimated that as many as 1,000,000 Americans areafflicted with inflammatory bowel disease, with male and female patientsappearing to be equally affected. Most cases are diagnosed before age30, but the disease can occur in the sixth, seventh, and later decadesof life.

IBS and chronic idiopathic constipation are pathological conditions thatcan cause a great deal of intestinal discomfort and distress but unlikethe inflammatory bowel diseases, IBS does not cause the seriousinflammation or changes in bowel tissue and it is not thought toincrease the risk of colorectal cancer. In the past, inflammatory boweldisease, celiac disease, and IBS were regarded as completely separatedisorders. Now, with the description of inflammation, albeit low-grade,in IBS, and of symptom overlap between IBS and celiac disease, thiscontention has come under question. Acute bacterial gastroenteritis isthe strongest risk factor identified to date for the subsequentdevelopment of postinfective irritable bowel syndrome. Clinical riskfactors include prolonged acute illness and the absence of vomiting. Agenetically determined susceptibility to inflammatory stimuli may alsobe a risk factor for irritable bowel syndrome. The underlyingpathophysiology indicates increased intestinal permeability andlow-grade inflammation, as well as altered motility and visceralsensitivity. Serotonin (5-hydroxytryptamine [5-HT]) is a key modulatorof gut function and is known to play a major role in pathophysiology ofIBS. The activity of 5-HT is regulated by cGMP.

While the precise causes of IBS and inflammatory bowel diseases (IBD)are not known, a disruption in the process of continual renewal of thegastrointestinal mucosa may contribute to disease pathology in IBD andaggravate IBS. The renewal process of the gastrointestinal lining is anefficient and dynamic process involving the continual proliferation andreplenishment of unwanted damaged cells. Proliferation rates of cellslining the gastrointestinal mucosa are very high, second only to thehematopoietic system. Gastrointestinal homeostasis depends on both theproliferation and programmed cellular death (apoptosis) of epithelialcells lining the gut mucosa. Cells are continually lost from the villusinto the lumen of the gut and are replenished at a substantially equalrate by the proliferation of cells in the crypts, followed by theirupward movement to the villus. The rates of cell proliferation andapoptosis in the gut epithelium can be increased or decreased in avariety of circumstances, e.g., in response to physiological stimulisuch as aging, inflammatory signals, hormones, peptides, growth factors,chemicals and dietary habits. In addition, an enhanced proliferationrate is frequently associated with a reduction in turnover time and anexpansion of the proliferative zone. The proliferation index is muchhigher in pathological states such as ulcerative colitis and othergastrointestinal disorders. Intestinal hyperplasia is a major promoterof gastrointestinal inflammation. Apoptosis and cell proliferationtogether regulate cell number and determine the proliferation index.Reduced rates of apoptosis are often associated with abnormal growth,inflammation, and neoplastic transformation. Thus, both increasedproliferation and/or reduced cell death may increase the proliferationindex of intestinal tissue, which may in turn lead to gastrointestinalinflammatory diseases.

In addition to a role for uroguanylin and guanylin as modulators ofintestinal fluid and ion secretion, these peptides may also be involvedin the continual renewal of gastrointestinal mucosa by maintaining thebalance between proliferation and apoptosis. For example, uroguanylinand guanylin peptides appear to promote apoptosis by controllingcellular ion flux. Given the prevalence of inflammatory conditions inWestern societies a need exists to improve the treatment options forinflammatory conditions, particularly of the gastrointestinal tract.

Peptide agonists of guanylate cyclase C agonists (“GCC agonists”) aredescribed in U.S. Pat. Nos. 7,041,786, 7,799,897, and U.S. PatentApplication Publication Nos. US2009/0048175, US 2010/0069306, US2010/0120694, US 2010/0093635, and US 2010/0221329. However, the solidphase synthesis of peptides for pharmaceutical application presents anumber of special problems such as an overall low yield (e.g., less than10%).

SUMMARY OF THE INVENTION

The present invention provides a process of preparing a peptide,particularly a peptide comprising the sequence of a peptide agonist ofguanylate cyclase C (“GCC”). The GCC agonist sequence is selected fromthe group consisting of SEQ ID NOs: 1-249. The GCC agonist sequence is namino acid units in length, with the N-terminal unit at position 1 andthe C-terminal unit at position n.

The process of the invention includes solid phase and/or solution phasesyntheses of suitable peptide fragments, subsequent fragmentcondensation in a solution to form a linear crude peptide, and optionaloxidative cyclization of cysteine amino acid residues of the linearcrude peptide to form the cyclized final product. Particularly, theprocess includes the following steps:

-   -   providing a first fragment having a first sequence of amino acid        units from position j through position k of the GCC agonist        sequence, wherein j is an integer between 1 and n−1, k is an        integer between 2 and n and is greater than j, and the first        fragment is protected except for an amino group of the amino        acid unit at position j, or alternatively, a carboxyl group of        the amino acid unit at position k,        -   providing a second fragment having a second sequence of            amino acid units from position h through position j−1 of the            GCC agonist sequence or having a third sequence of amino            acid units from position k+1 through position m of the GCC            agonist sequence, wherein h is an integer between 1 and n−2            and is smaller than j, m is an integer between k+2 and n,            and the second fragment is protected except for a carboxyl            group of the amino acid unit at position j−1 or an amino            group of the amino acid unit at position k+1, and        -   coupling the first and the second fragments via a            solution-phase synthesis to yield a protected peptide having            a sequence of amino acid units from position h through            position k of the GCC agonist sequence or a protected            peptide having a sequence of amino acid units from position            j through position m of the GCC agonist sequence.

The process of the invention may include one or more of the featuresdescribed in the following embodiments.

In one embodiment, the GCC agonist sequence is selected from the groupconsisting of SEQ ID NOs: 1, 8, 9, 55, 56, 58, and 59.

In one embodiment, the GCC agonist sequence is SEQ ID NO: 1 or 9. In oneembodiment, h is 1, j is 7, and k is 16. More specifically, the firstfragment has a sequence of amino acid units from position 7 throughposition 16 of SEQ ID NO: 1 or 9 and the second fragment has a sequenceof amino acid units from position 1 through position 6 of SEQ ID NO: 1or 9. In another embodiment, h is 7, j is 15, and k is 16. Morespecifically, the first fragment has a sequence of amino acid units fromposition 15 through position 16 of SEQ ID NO: 1 or 9, the secondfragment has a sequence of amino acid units from position 7 throughposition 14 of SEQ ID NO: 1 or 9, and the protected peptide produced viacoupling the first and second fragments has a sequence of amino acidunits from position 7 through position 16 of SEQ ID NO: 1 or 9. In oneembodiment, the process of the invention further includes deprotectingan amino group of the amino acid unit at position 7 of the protectedpeptide having the sequence of amino acid units from position 7 throughposition 16 of SEQ ID NO: 1 or 9 to yield a position-7 reactive peptide.In one embodiment, the process of the invention further includesproviding a third fragment having a sequence of amino acid units fromposition 1 through position 6 of SEQ ID NO: 1 or 9, wherein the thirdfragment is protected except for a carboxyl group of the amino acid atposition 6. In one embodiment, the process further includes coupling thethird fragment and the position-7 reactive peptide via a solution-phasesynthesis to yield a protected linear peptide having a sequence of aminoacid units from position 1 through position 16 of SEQ ID NO: 1 or 9. Inone embodiment, the process further includes deprotecting the protectedlinear peptide to yield a deprotected linear peptide. In one embodiment,the process further comprises oxidizing the deprotected linear peptideto yield the peptide having the GCC agonist sequence of SEQ ID NOs: 1 or9.

In one embodiment, each of the first and second fragments is not morethan 10 amino acid units in length (e.g., 2-10 amino acid units inlength, 3-9 amino acid units in length, or 4-8 amino acid units inlength).

In one embodiment, the second fragment has the second sequence of aminoacid units from position h through position j−1 of the GCC agonistsequence and either one or both of the amino acid unit of the secondfragment at position j−1 and the amino acid unit of the first fragmentat position k is selected from the group consisting of glycine, proline,leucine, alanine, and arginine. In one embodiment, at least one of theamino acid units at positions j−1 and k is either glycine or proline.

In one embodiment, the second fragment has the third sequence of aminoacid units from position k+1 through position m of the GCC agonistsequence and either one or both of the amino acid unit of the thirdfragment at position m and the amino acid unit of the first fragment atposition k is selected from the group consisting of glycine, proline,leucine, alanine, and arginine. In one embodiment, at least one of theamino acid units at positions m and k is either glycine or proline.

In one embodiment, k is n. In one embodiment, the second fragment hasthe second sequence of amino acid units from position h through positionj−1 of the GCC agonist sequence, and the amino acid unit at positionsj−1 is selected from the group consisting of glycine, proline, leucine,alanine, and arginine. In one embodiment, the amino acid unit atposition j−1 is either glycine or proline. In one embodiment, theprocess of the invention further comprises deprotecting an amino groupof the amino acid unit at position h of the protected peptide having asequence of amino acid units from position h through position k of theGCC agonist sequence to yield a position-h reactive peptide. In oneembodiment, the process further comprises providing a fourth fragmenthaving a sequence of amino acid units from position 1 through positionh−1 of the GCC agonist sequence, wherein the fourth fragment isprotected except for a carboxyl group of the amino acid unit at positionh−1. In one embodiment, the fourth fragment is not more than 10 aminoacid units in length (e.g., 2-10 amino acid units in length, 3-9 aminoacid units in length, or 4-8 amino acid units in length). In oneembodiment, the process further includes coupling the fourth fragmentand the position-h reactive peptide via a solution-phase synthesis toyield a protected linear peptide having a sequence of amino acid unitsfrom position 1 through position n of the GCC agonist sequence. In oneembodiment, the process further comprises deprotecting the protectedlinear peptide to yield a deprotected linear peptide having a sequenceof amino acid units from position 1 through position n of the GCCagonist sequence. In one embodiment, the process of the inventionfurther comprises oxidizing the deprotected linear peptide to yield thepeptide comprising the GCC agonist sequence selected from the groupconsisting of SEQ ID NOs: 1-249.

In one embodiment, at least one of the fragments of the peptide ofinterest (e.g., the first, second, third, and/or fourth fragments) isprovided via a solid-phase peptide synthesis. In one embodiment, thesolid-phase peptide synthesis is a Fmoc solid-phase synthesis. In oneembodiment, the Fmoc solid-phase synthesis is performed on2-chlorotrityl resin, such as those having 1% DVB and a substitutionrate ranging from 0.3 mmol/g to 1.2 mmol/g (e.g., 0.9-1.1 mmol/g).

The methods of this invention unexpectedly produce GCC agonist peptidesof high purities (e.g., >96%) at high yields (e.g., >14%) compared toconventional step-by-step solid-phase peptide synthesis (SPPS), wherethe overall yield of the peptides having a 96% purity level isapproximately 5%. The fragment condensation process (i.e., hybridsolution- and solid-phase process) of this invention also requires muchless time for synthesizing GCC peptides than conventional SPPS. Inaddition to the cost effectiveness afforded by the increased yields andreduced time, the process of this invention is also readily scalable forcommercial production.

In another aspect, the invention features the peptides, e.g., GCCagonist peptides, prepared by the methods described herein.

In still another aspect, the invention also provides a process ofpurifying the peptide comprising the GCC agonist sequence selected fromthe group consisting of SEQ ID NOs: 1-249. The process comprisesadsorbing the peptide onto a polymeric adsorbent column, optionallyrinsing the peptide with deionized water, eluting the optionally rinsedpeptide off the polymeric adsorbent column with a solvent mixture (e.g.,an alcohol aqueous solution) to form a peptide solution, removing partor all of the solvent mixture (e.g., water and the alcohol) from thepeptide solution to precipitate the peptide, and optionally adding anether to the dewatered peptide to facilitate precipitation of thepeptide.

In one embodiment, the peptide is precipitated by concentrating thepeptide solution by removing water and the alcohol under vacuum withoutthe need of adding an ether. In another embodiment, an ether is added tofacilitate the precipitation, e.g., by hastening the precipitationprocess.

In one embodiment, rinsing the peptide after adsorption onto a polymericadsorbent column is not needed when, e.g., the peptide adsorbed issubstantially free of water-soluble salts (e.g., phosphates oracetates). In this context “substantially” free of water-soluble saltsmeans that the salt content of the peptide is preferably less than 5%,less than 4.5%, less than 4.25%, less than 4%, less than 3.5%, less than3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, lessthan 0.5%, less than 0.25%, or less than 0.1%, of the total weight ofthe peptide.

In one embodiment, the polymeric adsorbent column comprises apolystyrene resin. In particular, the resin is selected so that thepurified peptide eluted or desorbed is not less than 80% of the peptideamount adsorbed on the resin, e.g., not less than 85%, not less than90%, or not less than 95%. In one embodiment, the resin is formed ofcrosslinked polystyrene with an average pore diameter greater than 5 nm,e.g., about 6-8 nm, 10-15 nm, 15-20 nm, or 25-30 nm.

In one embodiment, the solvent mixture used for eluting the peptidecomprises water, e.g., a mixture of water and one or more secondsolvents which can form azeotrope with water such as ethanol,isopropanol, tert-butanol, 2-butanol, 1-chloro-2-propanol,1-methoxy-2-propanol, 2-methoxy-ethanol, 2-methyl-2-propanol, aceticacid, methyl acetate, and propyl acetate. The one or more secondsolvents can be Class 3 solvents (or low-toxicity solvents) as definedin the ICH guideline. In another embodiment, the solvent mixture usedfor eluting comprises ether, such as a mixture of ethanol/ether orisopropanol/ether

In one embodiment, the alcohol aqueous solution comprises isopropanol,propanol, tert-butanol, 2-butanol, or ethanol.

In one embodiment, the ether comprises diethyl ether or MTBE.

In one embodiment, the process further includes salt exchanging thepeptide by washing the peptide with an aqueous solution comprising anammonium salt (e.g., ammonium acetate), acetic acid, and/or an acetatesalt (e.g., sodium acetate) before the purification. In one embodiment,if the process includes the salt exchanging step, the process alsoincludes rinsing the peptide with deionized water after adsorption onthe polymeric resin column to desalt the peptide.

In one embodiment, the process further includes lyophilizing the peptideafter the salt exchanging step before the purification.

In one embodiment, the process further includes drying the precipitatedpeptide after adding the ether, e.g., under reduced pressure.

In one embodiment, the purified peptide is selected from the groupconsisting of SEQ ID NOs: 1, 8, 9, and 56. Preferably, the purifiedpeptide is SEQ ID NO: 1 or 9.

The purification methods of this invention unexpectedly produce GCCagonist peptides of high purities (e.g., >96%) and high bulk and/or tapdensities (e.g., >0.3 g/mL) compared to conventional purificationmethods such as lyophilization, where the bulk and/or tap densities ofthe purified are at approximately 10 times lower than those of thepeptides purified by the methods of this invention. The purificationprocess of this invention also requires much less time than conventionallyophilization, which can potentially lead to reductions in topoisomercontent, deamidation degradation products, and other impurities. Thepurification process of this invention is also readily scalable forcommercial production compared to conventional lyophilization.

In yet another aspect, the invention features the peptides, e.g., GCCagonist peptides, purified by the process described herein. The purifiedpeptides may have one or more of the following features.

In one embodiment, the purified peptide has a bulk density of no lessthan 0.05 g/mL, no less than 0.1 g/mL, no less than 0.2 g/mL, no lessthan 0.3 g/mL, no less than 0.4 g/mL, or no less than 0.5 g/mL. Forexample, the purified peptide has a bulk density ranging between 0.05g/mL and 2 g/mL.

In one embodiment, the purified peptide has a tap density of no lessthan 0.08 g/mL, no less than 0.1 g/mL, no less than 0.15 g/mL, no lessthan 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, no lessthan 0.5 g/mL, or no less than 0.6 g/mL. For example, the purifiedpeptide has a tap density ranging between 0.08 g/mL and 2 g/mL.

In one embodiment, the purified peptide has a chromatographic purity ofno less than 96%, no less than 97%, or no less than 98%. For example,the GCC agonist peptide has chromatographic impurity content of nogreater than 4%, no greater than 3.5%, no greater than 3%, no greaterthan 2.5%, no greater than 2%, no greater than 1.5%, or no greater than1%. The chromatographic impurity content is determined as total areapercentages of impurities by HPLC. The chromatographic impurity contentincludes topoisomer content. The impurities do not include anypharmaceutically acceptable excipient used for drug formulation.

In one embodiment, the purified peptide is substantially free ofcontaminants resulted from the peptide preparation process such asorganic solvents used in the process, e.g., ammonium, acetonitrile,acetamide, alcohol (e.g., methanol, ethanol, or isopropanol), TFA, etheror other contaminants. In this context “substantially” free ofcontaminants means that the contaminant content of the peptide at theend of the purification process is preferably less than 0.5%, less than0.3%, less than 0.25%, less than 0.1%, less than 0.05%, less than 0.04%,less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%,less than 0.003%, or less than 0.001% of the total weight of thepeptide. For example, the purified peptide contains <0.01% acetamide,<0.3% ammonium ion, <0.01% acetonitrile, and/or <0.1% TFA. The contentof contaminants can be determined by conventional methods such as gaschromatography. Preferably, the residual solvents in the purifiedpeptide of the invention are less than the limits set in the ICHguidelines, e.g., IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R5)(available athttp://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3C/Step4/Q3C_R5_Step4.pdf). For example, the purified peptide contains <410 ppmacetonitrile (e.g., <40 ppm or <20 ppm), <5000 ppm ethanol (e.g., <140ppm), <5000 ppm isopropanol, <5000 ppm ethyl acetate (e.g., <20 ppm),<3000 ppm methanol (e.g., <250 ppm), <5000 ppm MTBE (e.g., <20 ppm),<290 ppm hexane, and/or <5000 ppm heptane or pentane.

In one embodiment, the purified peptide is substantially free oftopoisomers. In this context “substantially” free of topoisomers meansthat the topoisomer content of the peptide at the end of thepurification process is preferably less than 2%, less than 1.5%, lessthan 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%,less than 0.2%, or less than 0.1%, of the total weight of the peptide.

In one embodiment, the purified peptide is substantially free of water.In this context “substantially” free of water means that the watercontent of the peptide at the end of the purification process ispreferably less than 7%, less than 6%, less than 5%, less than 4.5%,less than 4.25%, less than 4%, less than 3.5%, less than 3%, less than2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, lessthan 0.25%, or less than 0.1%, of the total weight of the peptide.

In one embodiment, the purified peptide includes the GCC agonistsequence of SEQ ID NO: 1.

In one embodiment, the purified peptide includes the GCC agonistsequence of SEQ ID NO: 9.

The invention also relates to a formulation (e.g., an oral formulation)containing the peptides prepared and/or purified by the methodsdescribed herein and in particular, a low dose formulation containing0.05-10 mg (e.g., 0.1 mg, 0.3 mg or 0.5 mg) of the purified peptides.The low-dose formulation can further have one or more additionalfeatures as described in PCT/US2011/051805 and can be prepared by themethods disclosed therein, such as dry blending.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing particle size distribution by sieving analysisfor lyophilized plecanatide and precipitated plecanatide.

FIG. 2 is an optical microscopic image of lyophilized plecanatide.

FIG. 3 is an optical microscopic image of precipitated plecanatide.

DETAILED DESCRIPTION

The invention provides processes of preparing peptides, e.g., peptideGCC agonists, in particular a hybrid solution- and solid-phase process.The process of the invention includes providing two or more fragments ofa peptide of interest via solid-phase and/or solution-phase synthesesand coupling them via a solution-phase synthesis to obtain the targetpeptide. The process may further include, if needed, oxidativecyclization of cysteine amino acid residues of a linear peptide formedby the fragment coupling to produce a cyclized peptide.

The fragments described above can be prepared by standard solution phasepeptide synthesis or solid phase peptide synthesis techniques in which apeptide linkage occurs through the direct condensation of the aminogroup (i.e., NH₂) of a first amino acid with the carboxy group (i.e.,COOH) of a second amino acid with the elimination of a water molecule.In one embodiment, at least one of the fragments is prepared by solidphase peptide synthesis.

Peptide bond synthesis by direct condensation, as formulated above,requires suppression of the reactive character of the amino group of thefirst and of the carboxyl group of the second amino acid. The maskingsubstituents (i.e., protecting groups) must permit their ready removal,without inducing breakdown of the labile peptide molecule. The term“protected peptide” or “protected peptide fragment” refers to a peptideor peptide fragment, in which all reactive groups on its constitutingamino acids, are masked by protecting groups, unless otherwisespecified. The term “deprotected peptide” or “deprotected peptidefragment” refers to a peptide or peptide fragment, in which all reactivegroups on its constituting amino acids, are free from being masked byprotecting groups, unless otherwise specified. The term “reactivegroups” refers to the groups forming the peptide bond and thoseinterfering with the peptide bond formation, such as amino, carboxyl,hydroxyl, and thiol (as in cysteine) groups. Examples of protectinggroups for amino include and are not limited to9-fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzoyl(Bz), acetyl (Ac), and benzyl (Bn). Examples of protecting groups forcarboxyl include trityl (triphenylmethyl, Trt) and O-tert-butyl (OtBu).Examples of protecting groups for thiol include acetamidomethyl (Acm),tert-butyl (tBu), 3-nitro-2-pyridine sulfenyl (NPYS),2-pyridine-sulfenyl (Pyr), and trityl (Trt). Additional examples ofprotecting groups are described in Greene, T. W., Wuts, P. G. M.,Protective Groups in Organic Synthesis, 3′ edition, John Wiley & Sons:New York, 1999, whose context is incorporated by reference herein.

In a preferred embodiment, the methods of the invention are used forpreparing SP-304 (plecanatide). In particular, three peptide fragments,A, B and C are prepared and then a linear peptide sequence is assembledby the condensation of fragment A, B and C as follows: preparingfragment A, Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-OH,by solid phase from 2-chloro-trityl chloride resin; preparing fragmentB, Fmoc-Cys(Acm)-Val-Asn-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-OH, by solidphase from 2-chlorotrityl chloride resin; preparing fragment C,Cys(Acm)-Leu-OtBu, by solution phase synthesis, coupling fragments B andC in solution phase to yield fragment B-C, and coupling fragments A andB-C to yield linear peptide A-B-C.

The side-chain-protected Fragments A (BocAA1-60H) and B (FmocAA7-140H)can be prepared by Fmoc SPPS using the super acid-sensitive2-chlorotrityl chloride (2-ClTrt) resin and Fmoc-protected amino acidderivatives, as shown in Scheme 1 below.

Fragment C (HAA15-16OtBu) can be prepared by the solution phasesynthesis and then be coupled to Fragment B (FmocAA7-140H) in solutionphase to give Fragment B-C (FmocAA7-16OtBu). The Fmoc protecting groupcan then be removed from Fragment B-C (FmocAA7-16OtBu) to giveHAA7-16OtBu, which is then coupled to Fragment A (BocAA1-60H) to yieldside-chain-protected linear SP-304 (BocAA1-16OtBu), as shown in Scheme 2below.

The side-chain-protected linear SP-304 (BocAA1-16OtBu) can be treatedwith trifluoroacetic acid/triisopropylsilane/ethanedithiol (TFA/TIS/EDT)to give the partially protected SP-304 (HAA1-160H) in which the 2 S-Acmgroups (as shown in Scheme 2 above) are intact. The partially protectedlinear SP-304 (HAA1-160H) can be oxidized by H₂O₂, followed bysimultaneous removal of the S-Acm groups and disulfide formation withiodine to give crude dicyclic SP-304, as shown in Schemes 3 and 4 below.

The solution of crude dicyclic SP-304 can then be purified andconcentrated, as shown in Scheme 3 above, by loading the solution to apolystyrenic adsorbent resin (e.g., D101 (Anhui Sanxing (China);crosslinked polystyrene; surface area 500-550 m²/g; average porediameter: 9-10 nm; pore volume: 1.18-1.24 ml/g; bulk density: 0.65-0.70g/ml; specific density: 1.03-1.07 g/ml; moisture: 67-75%; particle size:0.315˜1.25 mm ≥95%; effective diameter: 0.4˜0.7 mm; uniformitycoefficient: ≤1.6%), DA201C, DA201H, ADS-8, and ADS-5) column, elutingthe dicyclic SP-304 from the column with an eluent (e.g., a 90% ethanolaqueous solution), concentrating the collected SP-304 solution underreduced pressure, and precipitating SP-304 with methyl t-butyl ether(MTBE). The precipitate can then be collected by filtration orcentrifugation, dried under high vacuum to give SP-304 in solid form.

As illustrated in Scheme 4 above, the solution of crude dicyclic SP-304can also be purified directly on preparative HPLC C18 column withacetonitrile (ACN), methanol, and/or water in various buffer system. Thecrude dicyclic SP-304 can also be purified via other methods known to askilled person in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection, and coupling reactions must go to completionand the side-chain blocking groups must be stable throughout thesynthesis.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (See, Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful fragment selection is necessary to minimize racemization duringfragment condensation. For example, racemization is minimized whenfragments contain C-terminal Gly or Pro. Solubility considerations arealso a factor. Solid phase peptide synthesis uses an insoluble polymerfor support during organic synthesis. The polymer-supported peptidechain permits the use of simple washing and filtration steps instead oflaborious purifications at intermediate steps. Solid-phase peptidesynthesis may generally be performed according to the method ofMerrifield et al., J. Am. Chem. Soc., 1963, 85:2149, which involvesassembling a linear peptide chain on a resin support using protectedamino acids. Solid phase peptide synthesis typically utilizes either theBoc or Fmoc strategy, both of which are well known in the art.

The processes of the present invention can be used to make anypeptide-based GCC agonist known in the art, such as analogs ofuroguanylin, guanylin, lymphoguanylin, linaclotide, ST peptides, SP-304,and SP-333. Non-limiting examples of such analogs of uroguanylin,guanylin, lymphoguanylin, linaclotide, SP-304, SP-333, and bacterial STpeptides are described in Section 1.1 below. In certain embodiments, themethods are used to prepare a peptide consisting essentially of an aminoacid sequence selected from SEQ ID NOs: 1-249. In a preferredembodiment, the peptide thusly made consists essentially of an aminoacid sequence selected from SEQ ID NOs: 1, 8, 9, 55, and 56. The term“consists essentially of” refers to a peptide that is identical to thereference peptide in its amino acid sequence or to a peptide that doesnot differ substantially in terms of either structure or function fromthe reference peptide. A peptide differs substantially from thereference peptide if its primary amino acid sequence varies by more thanthree amino acids from the reference peptide or if its activation ofcellular cGMP production is reduced by more than 50% compared to thereference peptide. Preferably, substantially similar peptides differ byno more than two amino acids and not by more than about 25% with respectto activating cGMP production. In preferred embodiments, the GCC agonistmade by the methods of the invention is a peptide comprising at least 12amino acid residues, and most preferably comprising between 12 and 26amino acids.

In another embodiment, the process of the invention is used forpreparing SP-353, which is a bacterial ST peptide analog. The generalstrategy for the hybrid synthesis of SP-353 includes solid phase andsolution phase syntheses to produce suitable peptide fragments (seeSchemes 5 and 6 below), subsequent segment condensation to form thelinear crude peptide (see Scheme 7 below), and natural oxidative foldingto form the cyclized final product (see Scheme 7 below). The samestrategy can also be used to produce other ST peptide analogs (such asSP-354, linaclotide, etc.) of similar amino acid sequences shown inTable II.

In yet another embodiment, the process of the invention is used forpreparing SP-333. The general strategy for the hybrid synthesis ofSP-333 includes solid phase and solution phase syntheses to producesuitable peptide fragments (see Schemes 8 and 9 below), subsequentsegment condensation to form the linear crude peptide (see Scheme 9below), oxidative folding to form the cyclized final product,purification, and lyophilization (see Scheme 10 below).

In another aspect, the invention also provides a process of purifyingpeptides, e.g., peptide GCC agonists. The general strategy for purifyingpeptides, including those synthesized by the hybrid methods disclosedherein, include, e.g., the steps illustrated in Scheme 11 below. It isunderstood that certain steps in Scheme 11 may be repeated (e.g.,rinsing column with deionized water) or absent (e.g., salt exchange oralcohol removal after dewatering) to optimize the purification process.

In yet another aspect, this invention provides a purified peptide, e.g.,peptide GCC agonists, purified by the precipitation process describedherein. Preferably, the purified peptide is SP-304 (SEQ ID NO:1) orSP-333 (SEQ ID NO:9). In one embodiment, the purified SP-304 or SP-333has a bulk density of no less than 0.05 g/mL, no less than 0.1 g/mL, noless than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, or noless than 0.5 g/mL. In a preferred embodiment, the purified SP-304 orSP-333 has a bulk density of about 0.05-2 g/mL, about 0.2-0.7 g/mL,about 0.3-0.6 g/mL, or about 0.4-0.5 g/mL. In one embodiment, thepurified SP-304 or SP-333 has a tap density of no less than 0.08 g/mL,no less than 0.1 g/mL, no less than 0.15 g/mL, no less than 0.2 g/mL, noless than 0.3 g/mL, no less than 0.4 g/mL, no less than 0.5 g/mL, or noless than 0.6 g/mL. For example, the purified SP-304 or SP-333 has a tapdensity of 0.08-2 g/mL, about 0.4-0.9 g/mL, about 0.5-0.8 g/mL, or about0.6-0.7 g/mL. In one embodiment, the purified peptide SP-304 or SP-333contains <0.01% acetamide (e.g., <28 ppm), <0.3% ammonium ion (e.g.,<0.25%), <0.01% acetonitrile (e.g., <20 ppm), and/or <0.1% TFA (e.g.,<0.09%). In one embodiment, the purified peptide SP-304 or SP-333 has abulk density of 0.4-0.5 g/mL, has a tap density of 0.6-0.7 g/mL, andcontains <0.01% acetamide (e.g., <28 ppm), <0.3% ammonium ion (e.g.,<0.25%), <0.01% acetonitrile (e.g., <20 ppm), and/or <0.1% TFA (e.g.,<0.09%).

1.1 GCC Agonists

The GCC agonists prepared by the processes of the invention can bind toguanylate cyclase C and stimulate intracellular production of cGMP.Optionally, the GCC agonists induce apoptosis and inhibit proliferationof epithelial cells. The term, “guanylate cyclase C” refers to atransmembrane form of guanylate cyclase that acts as the intestinalreceptor for the heat-stable toxin (ST) peptides secreted by entericbacteria. Guanylate cyclase C is also the receptor for the naturallyoccurring peptides guanylin and uroguanylin. The possibility that theremay be different receptors for each of these peptides has not beenexcluded. Hence, the term “guanylate cyclase C” may also encompass aclass of transmembrane guanylate cyclase receptors expressed onepithelial cells lining the gastrointestinal mucosa.

The term “GCC agonist” refers to both peptides and non-peptide compoundssuch as that bind to an intestinal guanylate cyclase C and stimulate theintracellular production of cGMP. Where the GCC agonist is a peptide,the term encompasses biologically active fragments of such peptides andpro-peptides that bind to guanylate cyclase C and stimulate theintracellular production of cGMP.

1.1.1 GCC Agonist Peptides

The GCC agonists prepared by the methods of the invention are preferablypeptides. In some embodiments, the GCC agonist peptide is less than 30amino acids in length. In particular embodiments, the GCC agonistpeptide is less than or equal to 30, 25, 20, 15, 14, 13, 12, 11, 10, or5 amino acids in length. Examples of GCC agonist peptides for use in theformulations and methods of the invention include those described inU.S. Pat. Nos. 7,879,802 and 8,034,782, and U.S. Publication Nos. US2010-0069306 and US 2010-0120694, each of which is incorporated byreference herein in its entirety.

Specific examples of GCC agonist peptides that can be prepared by themethods of the invention include those described in Tables I-VII below.As used Tables I-VII, the terms “PEG3” or “3PEG” refer to a polyethyleneglycol such as aminoethyloxy-ethyloxy-acetic acid (AeeA), and polymersthereof. The term “X_(aa)” refers to any natural or unnatural amino acidor amino acid analogue. The term “M_(aa)” refers to a cysteine (Cys),penicillamine (Pen) homocysteine, or 3-mercaptoproline. The term“Xaa_(n1)” is meant to denote an amino acid sequence of any natural orunnatural amino acid or amino acid analogue that is one, two or threeresidues in length; Xaa_(n2) is meant to denote an amino acid sequencethat is zero or one residue in length; and Xaa_(n3) is meant to denotean amino acid sequence zero, one, two, three, four, five or six residuesin length. Additionally, any amino acid represented by Xaa, Xaa_(n1),Xaa_(n2), or Xaa_(n3) may be an L-amino acid, a D-amino acid, amethylated amino acid or any combination of thereof. Optionally, any GCCagonist peptide represented by Formulas I to XX in the tables maycontain one or more polyethylene glycol residues at the N-terminus,C-terminus or both.

In certain embodiments, a GCC agonist prepared by the methods of theinvention comprises a peptide selected from SEQ ID NOs: 1-249, thesequences of which are set forth below in Tables I to VII below. In oneembodiment, a GCC agonist prepared by the methods of the inventioncomprises the peptide designated by SEQ ID NOs: 1, 8, 9, 55, or 56.

In certain embodiments, a GCC agonist prepared by the methods of theinvention comprises a peptide that is substantially equivalent to apeptide selected from SEQ ID NOs: 1-249. The term “substantiallyequivalent” refers to a peptide that has an amino acid sequenceequivalent to that of the binding domain where certain residues may bedeleted or replaced with other amino acids without impairing thepeptide's ability to bind to an intestinal guanylate cyclase receptorand stimulate fluid and electrolyte transport.

In certain embodiments, the GCC agonist peptides are analogues ofuroguanylin or a bacterial ST peptide. Uroguanylin is a circulatingpeptide hormone with natriuretic activity. An ST peptide is a member ofa family of heat stable enterotoxins (ST peptides) secreted bypathogenic strains of E. coli and other enteric bacteria that activateguanylate cyclase receptor and cause secretory diarrhea. Unlikebacterial ST peptides, the binding of uroguanylin to guanylate cyclasereceptor is dependent on the physiological pH of the gut. Therefore,uroguanylin is expected to regulate fluid and electrolyte transport in apH dependent manner and without causing severe diarrhea.

The GCC agonist peptides prepared by the methods of the invention can bepolymers of L-amino acids, D-amino acids, or a combination of both. Forexample, in various embodiments, the peptides are D retro-inversopeptides. The term “retro-inverso isomer” refers to an isomer of alinear peptide in which the direction of the sequence is reversed andthe chirality of each amino acid residue is inverted. See, e.g., Jamesonet al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693(1994). The net result of combining D-enantiomers and reverse synthesisis that the positions of carbonyl and amino groups in each amide bondare exchanged, while the position of the side-chain groups at each alphacarbon is preserved. Unless specifically stated otherwise, it ispresumed that any given L-amino acid sequence of the invention may bemade into a D retro-inverso peptide by synthesizing a reverse of thesequence for the corresponding native L-amino acid sequence.

The GCC agonist peptides prepared by the methods of the invention areable to induce intracellular cGMP production in cells and tissuesexpressing guanylate cyclase C. In certain embodiments, the GCC agonistpeptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared to naturally occurring GCC agonists such asuroguanylin, guanylin, or ST peptides. Optionally, the GCC agonistpeptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared to SP-304 (SEQ ID NO:1). In furtherembodiments, the GCC agonist peptide stimulates apoptosis, e.g.,programmed cell death, or activate the cystic fibrosis transmembraneconductance regulator (CFTR).

In some embodiments, the GCC agonist peptides prepared by the methods ofthe invention are more stable than naturally occurring GCC agonistsand/or SP-304 (SEQ ID NO:1), SP-339 (linaclotide) (SEQ ID NO: 55) orSP-340 (SEQ ID NO: 56). For example, the GCC agonist peptide degrades2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 90% or less compared tonaturally occurring GCC agonists and/or SP-304, SP-339 (linaclotide) orSP-340. In certain embodiments, the GCC agonist peptides for use in theformulations and methods of the invention are more stable to proteolyticdigestion than naturally occurring GCC agonists and/or SP-304 (SEQ IDNO:1), SP-339 (linaclotide) (SEQ ID NO: 55) or SP-340 (SEQ ID NO: 56).In one embodiment, a GCC agonist peptide is pegylated in order to renderthe peptides more resistant towards proteolysis by enzymes of thegastrointestinal tract. In a preferred embodiment, the GCC agonistpeptide is pegylated with the aminoethyloxy-ethyloxy-acetic acid (Aeea)group at its C-terminal end, at its N-terminal end, or at both termini.

Specific examples of GCC agonist peptides that can be prepared by themethods of the invention include a peptide selected from the groupdesignated by SEQ ID NOs: 1-249.

In one embodiment, the GCC agonist peptide is a peptide having the aminoacid sequence of any one of Formulas X-XVII (e.g. SEQ ID NOs: 87-98).

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula I, wherein at least one amino acid ofFormula I is a D-amino acid or a methylated amino acid and/or the aminoacid at position 16 is a serine. Preferably, the amino acid at position16 of Formula I is a D-amino acid or a methylated amino acid. Forexample, the amino acid at position 16 of Formula I is a d-leucine or ad-serine. Optionally, one or more of the amino acids at positions 1-3 ofFormula I are D-amino acids or methylated amino acids or a combinationof D-amino acids or methylated amino acids. For example, Asn¹, Asp² orGlu³ (or a combination thereof) of Formula I is a D-amino acid or amethylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula I is a leucine, serine or tyrosine.

In alternative embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula II, wherein at least one amino acidof Formula II is a D-amino acid or a methylated amino acid. Preferably,the amino acid denoted by Xaa_(n2) of Formula II is a D-amino acid or amethylated amino acid. In some embodiments, the amino acid denoted byXaa_(n2) of Formula II is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more amino acids denoted by Xaa_(n1) ofFormula II is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position Xaa⁶ of Formula II is a leucine, a serine, or atyrosine.

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula III, wherein at least one amino acid ofFormula III is a D-amino acid or a methylated amino acid and/or Maa isnot a cysteine. Preferably, the amino acid denoted by Xaa_(n2) ofFormula III is a D-amino acid or a methylated amino acid. In someembodiments the amino acid denoted by Xaa_(n2) of Formula III is aleucine, a d-leucine, a serine, or a d-serine. Preferably, the one ormore amino acids denoted by Xaa_(n1) of Formula III is a D-amino acid ora methylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula III is a leucine, a serine, or a tyrosine.

In other embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula IV, wherein at least one amino acid ofFormula IV is a D-amino acid or a methylated amino acid, and/or Maa isnot a cysteine. Preferably, the Xaa_(n2) of Formula IV is a D-amino acidor a methylated amino acid. In some embodiments, the amino acid denotedby Xaa_(n2) of Formula IV is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more of the amino acids denoted byXaa_(n1) of Formula IV is a D-amino acid or a methylated amino acid.Preferably, the amino acid denoted Xaa⁶ of Formula IV is a leucine, aserine, or a tyrosine.

In further embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula V, wherein at least one amino acid ofFormula V is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid. For example, the amino acid at position 16 (i.e., Xaa¹⁶) ofFormula V is a d-leucine or a d-serine. Optionally, one or more of theamino acids at position 1-3 of Formula V are D-amino acids or methylatedamino acids or a combination of D-amino acids or methylated amino acids.For example, Asn′, Asp² or Glu³ (or a combination thereof) of Formula Vis a D-amino acids or a methylated amino acid. Preferably, the aminoacid denoted at Xaa⁶ of Formula V is a leucine, a serine, or a tyrosine.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula VI, VII, VIII, or IX. Preferably, theamino acid at position 6 of Formula VI, VII, VIII, or IX is a leucine, aserine, or a tyrosine. In some aspects the amino acid at position 16 ofFormula VI, VII, VIII, or IX is a leucine or a serine. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI orXVII. Optionally, one or more amino acids of Formulas X, XI, XII, XIII,XIV, XV, XVI or XVII is a D-amino acid or a methylated amino acid.Preferably, the amino acid at the carboxy terminus of the peptidesaccording to Formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is aD-amino acid or a methylated amino acid. For example the amino acid atthe carboxy terminus of the peptides according to Formulas X, XI, XII,XIII, XIV, XV, XVI or XVII is a D-tyrosine.

Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine,phenylalanine or a serine. Most preferably the amino acid denoted byXaa⁶ of Formula XIV is a phenylalanine or a serine. Preferably, theamino acid denoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, aphenylalanine, or a serine. Most preferably, the amino acid positionXaa⁴ of Formula V, XVI or XVII is a phenylalanine or a serine.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula XVIII. Preferably, the amino acid at position 1of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine.Preferably, the amino acid at position 2 and 3 of Formula XVIII is aglutamic acid, or an aspartic acid. Preferably, the amino acid atposition 5 a glutamic acid. Preferably, the amino acid at position 6 ofFormula XVIII is an isoleucine, valine, serine, threonine, or tyrosine.Preferably, the amino acid at position 8 of Formula XVIII is a valine orisoleucine. Preferably, the amino acid at position 9 of Formula XVIII isan asparagine. Preferably, the amino acid at position 10 of FormulaXVIII is a valine or a methionine. Preferably, the amino acid atposition 11 of Formula XVIII is an alanine. Preferably, the amino acidat position 13 of Formula XVIII is a threonine. Preferably, the aminoacid at position 14 of Formula XVIII is a glycine. Preferably, the aminoacid at position 16 of Formula XVIII is a leucine, serine or threonine

In alternative embodiments, GCRA peptides include peptides containingthe amino acid sequence of Formula XIX. Preferably, the amino acid atposition 1 of Formula XIX is a serine or asparagine. Preferably, theamino acid at position 2 of Formula XIX is a histidine or an asparticacid. Preferably, the amino acid at position 3 of Formula XIX is athreonine or a glutamic acid. Preferably, the amino acid at position 5of Formula XIX is a glutamic acid. Preferably, the amino acid atposition 6 of Formula XIX is an isoleucine, leucine, valine, ortyrosine. Preferably, the amino acid at position 8, 10, 11, or 13 ofFormula XIX is an alanine. Preferably, the amino acid at position 9 ofFormula XIX is an asparagine or a phenylalanine. Preferably, the aminoacid at position 14 of Formula XIX is a glycine.

In further embodiments, GCRA peptides include peptides containing theamino acid sequence of Formula XX. Preferably, the amino acid atposition 1 of Formula XX is a glutamine. Preferably, the amino acid atposition 2 or 3 of Formula XX is a glutamic acid or an aspartic acid.Preferably, the amino acid at position 5 of Formula XX is a glutamicacid. Preferably, the amino acid at position 6 of Formula XX isthreonine, glutamine, tyrosine, isoleucine, or leucine. Preferably, theamino acid at position 8 of Formula XX is isoleucine or valine.Preferably, the amino acid at position 9 of Formula XX is asparagine.Preferably, the amino acid at position 10 of Formula XX is methionine orvaline. Preferably, the amino acid at position 11 of Formula XX isalanine. Preferably, the amino acid at position 13 of Formula XX is athreonione. Preferably, the amino acid at position 1 of Formula XX is aglycine. Preferably, the amino acid at position 15 of Formula XX is atyrosine. Optionally, the amino acid at position 15 of Formula XX is twoamino acid in length and is Cysteine (Cys), Penicillamine (Pen)homocysteine, or 3-mercaptoproline and serine, leucine or threonine.

In certain embodiments, one or more amino acids of the GCC agonistpeptides are replaced by a non-naturally occurring amino acid or anaturally or non-naturally occurring amino acid analog. Such amino acidsand amino acid analogs are known in the art. See, for example, Hunt,“The Non-Protein Amino Acids,” in Chemistry and Biochemistry of theAmino Acids, Barrett, Chapman, and Hall, 1985. In some embodiments, anamino acid is replaced by a naturally-occurring, non-essential aminoacid, e.g., taurine. Non-limiting examples of naturally occurring aminoacids that can be replaced by non-protein amino acids include thefollowing: (1) an aromatic amino acid can be replaced by3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine,L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr); (2) Phg andnorTyr and other amino acids including Phe and Tyr can be substitutedby, e.g., a halogen, —CH3, —OH, —CH2NH3, —C(O)H, —CH2CH3, —CN,—CH2CH2CH3, —SH, or another group; (3) glutamine residues can besubstituted with gamma-Hydroxy-Glu or gamma-Carboxy-Glu; (4) tyrosineresidues can be substituted with an alpha substituted amino acid such asL-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr;Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala;beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala;beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp; (5) prolineresidues can be substituted with homopro (L-pipecolic acid);hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or anN(alpha)-C(alpha) cyclized amino acid analogues with the structure: n=0,1, 2, 3; and (6) alanine residues can be substituted withalpha-substituted or N-methylated amino acid such as alpha-aminoisobutyric acid (aib), L/D-alpha-ethylalanine (LID-isovaline),L/D-methylvaline, or L/D-alpha-methylleucine or a non-natural amino acidsuch as beta-fluoro-Ala. Alanine can also be substituted with: n=0, 1,2, 3 Glycine residues can be substituted with alpha-amino isobutyricacid (aib) or L/D-alpha-ethylalanine (L/D-isovaline).

Further examples of non-natural amino acids include: an unnatural analogof tyrosine; an unnatural analogue of glutamine; an unnatural analogueof phenylalanine; an unnatural analogue of serine; an unnatural analogueof threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; an amino acid that is amidated at a site thatis not naturally amidated, a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g., a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an α, α disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; aρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc β-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; anisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue; a dopa, O-methyl-L-tyrosine;a glycosylated amino acid; a p-(propargyloxy)phenylalanine;dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine;3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);ε-Acetyl-Lysine; β-alanine; aminobenzoyl derivative; aminobutyric acid(Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AM);cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine;nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindolecarboxylate; ornithine (Orn); penicillamine (PEN);tetrahydroisoquinoline; acetamidomethyl protected amino acids andpegylated amino acids. Further examples of unnatural amino acids andamino acid analogs can be found in U.S. 20030108885, U.S. 20030082575,US20060019347 (paragraphs 410-418) and the references cited therein. Thepolypeptides of the invention can include further modificationsincluding those described in US20060019347, paragraph 589. Exemplary GCCagonist peptides which include a non-naturally occurring amino acidinclude for example SP-368 and SP-369.

In some embodiments, the GCC agonist peptides are cyclic peptides. GCCagonist cyclic peptides can be prepared by methods known in the art. Forexample, macrocyclization is often accomplished by forming an amide bondbetween the peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains, such as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39:51-124 (1988). In various embodiments, the GCC agonist peptides are[4,12; 7,15] bicycles.

In certain embodiments, one or both Cys residues which normally form adisulfide bond in a GCC agonist peptide are replaced with homocysteine,penicillamine, 3-mercaptoproline (Kolodziej et al. 1996 Int. J. Pept.Protein Res. 48:274), β, β dimethylcysteine (Hunt et al. 1993 Int. J.Pept. Protein Res. 42:249), or diaminopropionic acid (Smith et al. 1978J. Med. Chem. 2 1:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds.

In certain embodiments, one or more disulfide bonds in a GCC agonistpeptide are replaced by alternative covalent cross-links, e.g., an amidelinkage (—CH₂CH(O)NHCH₂— or —CH₂NHCH(O)CH₂—), an ester linkage, athioester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage,a thiourea linkage, a phosphonate ester linkage, an alkyl linkage(—CH₂CH₂CH₂CH₂—), an alkenyl linkage (—CH₂CH═CHCH₂—), an ether linkage(—CH₂CH₂OCH₂— or —CH₂OCH₂CH₂—), a thioether linkage (—CH₂CH₂SCH₂— or—CH₂SCH₂CH₂—), an amine linkage (—CH₂CH₂NHCH₂— or —CH₂NHCH₂CH₂—) or athioamide linkage (—CH₂C(S)NHCH₂— or —CH₂NHC (S)CH₂—). For example, Leduet al. (Proc. Natl. Acad. Sci. 100:11263-78, 2003) describe methods forpreparing lactam and amide cross-links. Exemplary GCC agonist peptideswhich include a lactam bridge include, for example, SP-370.

In certain embodiments, the GCC agonist peptides have one or moreconventional polypeptide bonds replaced by an alternative bond. Suchreplacements can increase the stability of the polypeptide. For example,replacement of the polypeptide bond between a residue amino terminal toan aromatic residue (e.g. Tyr, Phe, Trp) with an alternative bond canreduce cleavage by carboxy peptidases and may increase half-life in thedigestive tract. Bonds that can replace polypeptide bonds include: aretro-inverso bond (C(O)—NH instead of NH—C(O); a reduced amide bond(NH—CH₂); a thiomethylene bond (S—CH₂ or CH₂—S); an oxomethylene bond(O—CH₂ or CH₂—O); an ethylene bond (CH₂—CH₂); a thioamide bond(C(S)—NH); a trans-olefine bond (CH═CH); a fluoro substitutedtrans-olefine bond (CF═CH); a ketomethylene bond (C(O)—CHR or CHR—C(O)wherein R is H or CH₃; and a fluoro-ketomethylene bond (C(O)—CFR orCFR—C(O) wherein R is H or F or CH₃.

In certain embodiments, the GCC agonist peptides are modified usingstandard modifications. Modifications may occur at the amino (N—),carboxy (C—) terminus, internally or a combination of any of thepreceding. In one aspect described herein, there may be more than onetype of modification on the polypeptide. Modifications include but arenot limited to: acetylation, amidation, biotinylation, cinnamoylation,farnesylation, formylation, myristoylation, palmitoylation,phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation,sulfurylation and cyclisation (via disulfide bridges or amidecyclisation), and modification by Cys3 or Cys5. The GCC agonist peptidesdescribed herein may also be modified by 2, 4-dinitrophenyl (DNP),DNP-lysine, modification by 7-Amino-4-methyl-coumarin (AMC),flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide,rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid),dabcyl, dabsyl, dansyl, texas red, FMOC, and Tamra(Tetramethylrhodamine). The GCC agonist peptides described herein mayalso be conjugated to, for example, polyethylene glycol (PEG); alkylgroups (e.g., C1-C20 straight or branched alkyl groups); fatty acidmoieties; combinations of PEG, alkyl groups and fatty acid moieties(See, U.S. Pat. No. 6,309,633; Soltero et al., 2001 Innovations inPharmaceutical Technology 106-110); BSA and KLH (Keyhole LimpetHemocyanin). The addition of PEG and other polymers which can be used tomodify polypeptides of the invention is described in US20060 19347section IX.

A GCC agonist peptide can also be a derivative of a GCC agonist peptidedescribed herein. For example, a derivative includes hybrid and modifiedforms of GCC agonist peptides in which certain amino acids have beendeleted or replaced. A modification may also include glycosylation.Preferrably, where the modification is an amino acid substitution, it isa conservative substitution at one or more positions that are predictedto be non-essential amino acid residues for the biological activity ofthe peptide. A “conservative substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

In one embodiment, a GCC agonist peptide prepared by the methodsdescribed herein is subjected to random mutagenesis in order to identifymutants having biological activity.

In one embodiment, the methods of the invention can be used to prepare aGCC agonist peptide that is substantially homologous to a GCC agonistpeptide described herein. Such substantially homologous peptides can beisolated by virtue of cross-reactivity with antibodies to a GCC agonistpeptide described herein.

Further examples of GCC agonist peptides that can be prepared by themethods of the invention are found in Tables I-VII below.

1.1.2 Alternative Preparation Methods of GCC Agonist Peptides and theirFragments

GCC agonist peptides and their fragments can be prepared using artrecognized techniques such as molecular cloning, peptide synthesis, orsite-directed mutagenesis.

In addition to the conventional solution- or solid-phase peptidesynthesis described above, the GCC agonist peptides or their fragmentscan be produced by modern cloning techniques. For example, the GCCagonist peptides are produced either in bacteria including, withoutlimitation, E. coli, or in other existing systems for polypeptide orprotein production (e.g., Bacillus subtilis, baculovirus expressionsystems using Drosophila Sf9 cells, yeast or filamentous fungalexpression systems, mammalian cell expression systems), or they can bechemically synthesized. If the GCC agonist peptide or variant peptide isto be produced in bacteria, e.g., E. coli, the nucleic acid moleculeencoding the polypeptide may also encode a leader sequence that permitsthe secretion of the mature polypeptide from the cell. Thus, thesequence encoding the polypeptide can include the pre sequence and thepro sequence of, for example, a naturally-occurring bacterial STpolypeptide. The secreted, mature polypeptide can be purified from theculture medium.

The sequence encoding a GCC agonist peptide described herein can beinserted into a vector capable of delivering and maintaining the nucleicacid molecule in a bacterial cell. The DNA molecule may be inserted intoan autonomously replicating vector (suitable vectors include, forexample, pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleicacid may be a bacterial or bacteriophage DNA such as bacteriophagelambda or M13 and derivatives thereof. Construction of a vectorcontaining a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include but are not limited to, E. coli, B. subtilis, Pseudomonas,Salmonella. The genetic construct also includes, in addition to theencoding nucleic acid molecule, elements that allow expression, such asa promoter and regulatory sequences. The expression vectors may containtranscriptional control sequences that control transcriptionalinitiation, such as promoter, enhancer, operator, and repressorsequences.

A variety of transcriptional control sequences are well known to thosein the art. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, or an internal ribosome entry site). The vector can becapable of autonomous replication or it can integrate into host DNA toensure stability during polypeptide production.

The protein coding sequence that includes a GCC agonist peptidedescribed herein can also be fused to a nucleic acid encoding apolypeptide affinity tag, e.g., glutathione S-transferase (GST), maltoseE binding protein, protein A, FLAG tag, hexa-histidine, myc tag or theinfluenza HA tag, in order to facilitate purification. The affinity tagor reporter fusion joins the reading frame of the polypeptide ofinterest to the reading frame of the gene encoding the affinity tag suchthat a translational fusion is generated. Expression of the fusion generesults in translation of a single polypeptide that includes both thepolypeptide of interest and the affinity tag. In some instances whereaffinity tags are utilized, DNA sequence encoding a protease recognitionsite will be fused between the reading frames for the affinity tag andthe polypeptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the GCC agonist peptides and variants described hereinin protein expression systems other than bacteria, and well known tothose skilled in the art, can also be used to produce polypeptides in abiological system.

The peptides disclosed herein may be modified by attachment of a secondmolecule that confers a desired property upon the peptide, such asincreased half-life in the body, for example, pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

TABLE I GCRA Peptides (SP-304 and Derivatives) SEQ Position of ID NameDisulfide bonds Structure NO SP-304 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶1 SP-326 C3: C11, C6: C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵2 SP-327 C2: C10, C5: C13Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴3 SP-328 C2: C10, C5: C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴4 SP-329 C2: C10, C5: C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 5SP-330 C1: C9, C4: C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asd-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²-Leu¹³ 6SP-331 C1: C9, C4: C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asd-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹² 7 SP332C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶8 SP-333 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶9 SP-334 C4: C12, C7: C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶10 SP-335 C4: C12, C7: C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶11 SP-336 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶12 SP-337 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶13 SP-338 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵14 SP-342 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG315 SP-343 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr13 -Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG316 SP-344 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Var-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG317 SP-347 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG318 SP-348 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶19 SP-350 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val10 Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶20 SP-352 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG321 SP-358 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-22 PEG3 SP-359 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶23 SP-360 C4: C12, C7: C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG324 SP-361 C4: C12, C7: C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ -PEG325 SP-362 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶26 SP-368 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dNal¹⁶27 SP-369 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶28 SP-370 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val0-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Orn¹⁵-dLeu¹29 SP-371 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶30 SP-372 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶31 N1 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ -PEG332 N2 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶33 N3 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ PEG334 N4 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ -PEG335 N5 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val10 Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶36 N6 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ -PEG337 N7 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val10 Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶38 N8 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG339 N9 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶40 N10 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG341 N11 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶ -PEG342 N12 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶43 N13 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶ -PEG344 Formula I C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶45 Formula II C4: C12, C7: C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaal Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa_(n2)¹⁶ 46 Formula 4: 12, 7: 15Xaa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴-Maa¹⁵- Xaa_(n2)47 III Formula 4: 12, 7: 15Xaa_(n1) - Maa⁴-Xaa⁵-Xaa⁶- Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹- Maa¹²-Xaa¹³-Xaa¹⁴- Maa¹⁵-Xaa¹⁶48 IV Formula V C4: C12, C7: C15Asn¹-Asp²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶49 Formula C4: C12, C7: C15dAsn¹-Glu²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-X3⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶50 VI Formula C4: C12, C7: C15dAsn¹-dGlu²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶51 VII Formula C4: C12, C7: C15dAsn¹-dAsp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶52 VII Formula C4: C12, C7: C15dAsn¹-dAsp²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶53 VIII Formula C4: C12, C7: C15dAsn¹-dGlu²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶54 IX

TABLE II Linaclotide and Derivatives Position of Disulfide SEQ ID Namebonds Structure NO: SP-339 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴55 (linaclotide) SP-340 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 56SP-349 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG357 SP-353 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶58 SP-354 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶59 SP-355 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-dTyr¹⁴60 SP-357 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴61 SP-374 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶62 SP-375 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶63 SP-376 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶64 SP-377 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶65 SP-378 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶66 SP-379 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶67 SP-380 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶68 SP-381 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶69 SP-382 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶70 SP-383 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶71 SP-384 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG372 N14 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG373 N15 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³74 N16 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG375 N17 C3: C8, C4: C12, C7: 15PEG3- Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-76 Tyr¹⁶-PEG3 N18 C3: C8, C4: C12, C7: 15PEG3- Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-77 Tyr¹⁶ N19 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-78 PEG3 N20 C3: C8, C4: C12, C7: 15PEG3- Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-79 Tyr¹⁶-PEG3 N21 C3: C8, C4: C12, C7: 15PEG3- Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-80 Tyr¹⁶ N22 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-81 PEG3 N23 C3: C8, C4: C12, C7: 15PEG3- Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-82 Tyr¹⁶-PEG3 N24 C3: C8, C4: C12, C7: 15PEG3- Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-83 Tyr¹⁶ N25 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-84 PEG3 N26 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴85 N27 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴86 N28 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³- 87N29 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 88N30 1: 6, 2: 10, 5: 13Pen¹-Pen²-Glu³-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³-Tyr¹⁴89 N31 1: 6, 2: 10, 5: 13Pen¹-Pen²-Glu³-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³ 90Formula X C9: C14 , C10: C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶- Asn⁷- Tyr⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Tyr¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-Xaa¹⁶-91 C13: 21 Xaa¹⁷-Cys¹⁸- Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIC9: C14, C10: C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷- Phe⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Phe¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-Xaa¹⁶-92 C13: 21 Xaa¹⁷-Cys¹⁸- Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIIC3: C8, C4: C12, C7: 15Asn¹- Phe²-Cys³-Cys4 - Xaa⁵-Phe⁶-Cys⁷-Cys8 - Xaa⁹-Xaa¹⁰- Xaa¹¹-Cys¹²- Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶93 Formula 3: 8, 4: 12, C: 15Asn¹- Phe²-Pen³-Cys4 - Xaa⁵-Phe⁶-Cys⁷-Pen8 - Xaa⁹-Xaa¹⁰- Xaa¹¹-Cys¹²- Xaa¹³-Xaa¹⁴-Cys¹⁵-94 XIII Xaa¹⁶ Formula 3: 8, 4: 12, 7: 15Asn¹- Phe²-Maa³-Maa4 - Xaa⁵-Xaa⁶-Maa⁷-Maa8 - Xaa⁹-Xaa¹⁰- Xaa¹¹-Maa¹²- Xaa¹³-Xaa¹⁴-Maa¹⁵-95 XIV Xaa¹⁶ Formula XV 1: 6, 2: 10, 5: 13Maa¹-Maa²-Glu³-Xaa⁴- Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-Tyr¹⁴96 Formula 1: 6, 2: 10, 5: 13Maa¹-Maa²-Glu³-Xaa⁴- Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-97 XVI Formula 1: 6, 2: 10, 5: 13Xaa_(n3)-Maa¹-Maa²-Xaa³-Xaa⁴-Maa⁵-Maa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Maa¹⁰-Xaa¹¹-Xaa¹²-Maa¹³-Xaa^(n2 )98 XVII

TABLE III GCRA Peptides Position of SEQ Disulfide ID Name bondsStructure NO: SP-363 C4: C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-99 C7: C15 AMIDE¹⁶ SP-364 C4: C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶100 C7: C15 SP-365 C4: C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer-101 C7: C15 AMIDE¹⁶ SP-366 C4: C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶102 C7: C15 SP-367 C4: C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr-103 C7: C15 AMIDE¹⁶ SP-373 C4: C12,Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-104 C7: C15 AMIDE¹⁶ SP-304 C4: C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-105 di PEG C7: C15 Leu¹⁶-PEG3 SP-304 C4: C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-106 N-PEG C7: C15 Leu¹⁶ SP-304 C4: C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶-107 C-PEG C7: C15 PEG3

TABLE IV SP-304 Analogs, Uroguanylin, and Uroguanylin Analogs Positionof SEQ Disulfide ID Name bonds Structure NO Formula C4: C12,Xaa¹- Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa¹⁶108 XVIII C7: C15 Urogua- C4: C12,Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶109 nylin C7: C15 N32 C4: C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶110 C7: C15 N33 C4: C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶111 C7: C15 N34 C4: C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶112 C7: C15 N35 C4: C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶113 C7: C15 N36 C4: C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶114 C7: C15 N37 C4: C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶115 C7: C15 N38 C4: C12,Asp¹-Glu²-Asp³-Cy⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶116 C7: C15 N39 C4: C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶117 C7: C15 N40 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶118 C7: C15 N41 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶119 C7: C15 N42 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶120 C7: C15 N43 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶121 C7: C15 N44 C4: C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶122 C7: C15 N45 C4: C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶123 C7: C15 N46 C4: C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶124 C7: C15 N47 C4: C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶125 C7: C15 N48 C4: C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶126 C7: C15 N49 C4: C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶127 C7: C15 N50 C4: C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶128 C7: C15 N51 C4: C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶129 C7: C15 N52 C4: C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶130 C7: C15 N53 C4: C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶131 C7: C15 N54 C4: C12,Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶132 C7: C15 N55 C4: C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶133 C7: C15 N56 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶134 C7: C15 N57 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶135 C7: C15 N58 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶136 C7: C15 N59 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶137 C7: C15 N60 C4: C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶138 C7: C15 N61 C4: C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶139 C7: C15 N62 C4: C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶140 C7: C15 N63 C4: C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶141 C7: C15 N65 C4C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶142 C7: C15 N66 C4: C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶143 C7: C15 N67 C4: C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶144 C7: C15 N68 C4: C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶145 C7: C15 N69 C4: C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶146 C7: C15 N70 C4: C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶147 C7: C15 N71 C4: C12,Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶148 C7: C15 N72 C4: C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶149 C7: C15 N73 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶150 C7: C15 N74 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶151 C7: C15 N75 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶152 C7: C15 N76 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶153 C7: C15 N77 C4: C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶154 C7: C15 N78 C4: C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶155 C7: C15 N79 C4: C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶156 C7: C15 N80 C4: C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶157 C7: C15 N81 C4: C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶158 C7: C15 N82 C4: C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶159 C7: C15 N83 C4: C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶160 C7: C15 N84 C4: C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶161 C7: C15 N85 C4: C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶162 C7: C15 N86 C4: C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶163 C7: C15 N87 C4: C12,Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶164 C7: C15 N88 C4: C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶165 C7: C15 N89 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶166 C7: C15 N90 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶167 C7: C15 N91 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶167 C7: C15 N92 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶169 C7: C15 N93 C4: C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶170 C7: C15 N94 C4: C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶171 C7: C15 N95 C4: C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶172 C7: C15 N96 C4: C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶173 C7: C15

TABLE V Guanylin and Analogs SEQ Position of ID Name Disulfide bondsStructure NO Formula 4: 12, 7: 15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵174 XIX Guany- C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Phe⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵175 lin N97 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵176 N98 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵177 N99 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵178 N100 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵179 N101 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵180 N102 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵181 N103 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵182 N104 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵183 N105 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵184 N106 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵185 N107 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵186 N108 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵187 N109 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵188 N110 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵189 N111 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵190 N112 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵191 N113 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵192 N114 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵193 N115 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵194 N116 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵195 N117 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵196 N118 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵197 N119 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵198 N120 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵199 N121 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵200 N122 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵201 N123 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵202 N124 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵203 N125 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵204 N126 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵205 N127 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵206 N128 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵207

TABLE VI Lymphoguanylin and Analogs Position of SEQ Disulfide ID Namebonds Structure NO Formula XX 4: 12,Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Xaa_(n1)¹⁵ 208 7: 15 Lymphogua- C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵209 nylin N129 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵210 N130 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵211 N131 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵212 N132 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵213 N133 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵214 N134 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵215 N135 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵216 N136 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵217 N137 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵218 N138 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵219 N139 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵220 N140 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵221 N141 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵222 N142 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵223 N143 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵224 N144 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵225 N145 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-226 C7: C15 Ser¹⁶ N146 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-227 C7: C15 Ser¹⁶ N147 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-228 C7: C15 Ser¹⁶ N148 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-229 C7: C15 Ser¹⁶ N149 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-230 C7: C15 Ser¹⁶ N150 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-231 C7: C15 Ser N151 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-232 C7: C15 Ser¹⁶ N152 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-233 C7: C15 Ser¹⁶ N153 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-234 C7: C15 Ser¹⁶ N154 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-235 C7: C15 Ser¹⁶ N155 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-236 C7: C15 Ser¹⁶ N156 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-237 C7: C15 Ser¹⁶ N157 C4: C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-238 C7: C15 Ser¹⁶ N158 C4: C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-239 C7: C15 Ser¹⁶ N159 C4: C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-240 C7: C15 Ser¹⁶ N160 C4: C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-241 C7: C15 Ser¹⁶

TABLE VII ST Peptide and Analogues Position of SEQ Disulfide ID Namebonds Structure NO ST C3: C8,Asn¹-Ser²-Ser³-Asn⁴-Ser⁵-Ser⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Glu¹¹-Lys¹²-Cys¹³-Cys¹⁴-242 Peptide C4: C12, Asn¹⁵-Pro¹⁶-Ala¹⁷-Cys¹⁸-Thr¹⁹-Gly²⁰-Cys²¹-Tyr²²C7: 15 N161 C3: C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-243 C4: C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 C7: 15 N162 C3: C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-244 C4: C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶ C7: 15 N163 C3: C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-245 C4: C12, Cys¹⁵-Tyr¹⁶-PEG3 C7: 15 N164 C3: C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-246 C4: C12, Cys¹⁵-Tyr¹⁶ C7: 15 N165 C3: C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-247 C4: C12, Cys¹⁵-dTyr¹⁶ C7: 15 N166 C3: C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-248 C4: C12, Cys¹⁵-dTyr¹⁶ C7: 15 N167 C3: C8, dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-249 C4: C12, Cys¹⁵-Tyr¹⁶ C7: 15

1.2 Methods of Use

The invention provides methods for treating or preventinggastrointestinal disorders and increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist or a formulation thereof to the subject. Non-limiting examplesof gastrointestinal disorders that can be treated or prevented accordingto the methods of the invention include irritable bowel syndrome (IBS),non-ulcer dyspepsia, H. pylori infection related ulcers, chronicintestinal pseudo-obstruction, functional dyspepsia, colonicpseudo-obstruction, duodenogastric reflux, gastroesophageal refluxdisease (GERD), ileus (e.g., postoperative ileus), gastroparesis,heartburn (high acidity in the GI tract), constipation (e.g.,constipation associated with use of medications such as opioids,osteoarthritis drugs, or osteoporosis drugs); post surgicalconstipation, constipation associated with neuropathic disorders,Crohn's disease, and ulcerative colitis.

In one embodiment, the invention provides methods for treating orpreventing gastrointestinal motility disorder, irritable bowel syndrome,a functional gastrointestinal disorder, gastroesophageal reflux disease,duodenogastric reflux, functional heartburn, dyspepsia, functionaldyspepsia, non-ulcer dyspepsia, gastroparesis, chronic intestinalpseudo-obstruction, colonic pseudo-obstruction, obesity, congestiveheart failure, or benign prostatic hyperplasia.

In one embodiment, the invention provides methods for treating orpreventing constipation and/or increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist or a formulation thereof to the subject. Clinically acceptedcriteria that define constipation range from the frequency of bowelmovements, the consistency of feces and the ease of bowel movement. Onecommon definition of constipation is less than three bowel movements perweek. Other definitions include abnormally hard stools or defecationthat requires excessive straining (Schiller 2001 Aliment Pharmacol Ther15:749-763). Constipation may be idiopathic (functional constipation orslow transit constipation) or secondary to other causes includingneurologic, metabolic or endocrine disorders. These disorders includediabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia,Multiple sclerosis, Parkinson's disease, spinal cord lesions,Neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprungdisease and cystic fibrosis. Constipation may also be the result ofsurgery or due to the use of drugs such as analgesics (like opioids),antihypertensives, anticonvulsants, antidepressants, antispasmodics andantipsychotics.

In various embodiments, the constipation is associated with use of atherapeutic agent; the constipation is associated with a neuropathicdisorder; the constipation is postsurgical constipation; theconstipation is associated with a gastrointestinal disorder; theconstipation is idiopathic (functional constipation or slow transitconstipation); the constipation is associated with neuropathic,metabolic or endocrine disorder (e.g., diabetes mellitus,hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis,Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomicneuropathy, Chagas disease, Hirschsprung disease or cystic fibrosis).Constipation may also be the result of surgery or due to the use ofdrugs such as analgesics (e.g., opioids), antihypertensives,anticonvulsants, antidepressants, antispasmodics and antipsychotics.

In one embodiment, the invention provides methods for treating orpreventing chronic idiopathic constipation and increasinggastrointestinal motility in a subject in need thereof by administeringan effective amount of a GCC agonist or a formulation thereof to thesubject.

The term “treating” as used herein refers to a reduction, a partialimprovement, amelioration, or a mitigation of at least one clinicalsymptom associated with the gastrointestinal disorders being treated.The term “preventing” refers to an inhibition or delay in the onset orprogression of at least one clinical symptom associated with thegastrointestinal disorders to be prevented. The term “effective amount”as used herein refers to an amount that provides some improvement orbenefit to the subject. In certain embodiments, an effective amount isan amount that provides some alleviation, mitigation, and/or decrease inat least one clinical symptom of the gastrointestinal disorder to betreated. In other embodiments, the effective amount is the amount thatprovides some inhibition or delay in the onset or progression of atleast one clinical symptom associated with the gastrointestinal disorderto be prevented. The therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject. The term“subject” preferably refers to a human subject but may also refer to anon-human primate or other mammal preferably selected from among amouse, a rat, a dog, a cat, a cow, a horse, or a pig.

The invention also provides methods for treating gastrointestinal cancerin a subject in need thereof by administering an effective amount of aGCC agonist or a formulation thereof to the subject. Non-limitingexamples of gastrointestinal cancers that can be treated according tothe methods of the invention include gastric cancer, esophageal cancer,pancreatic cancer, colorectal cancer, intestinal cancer, anal cancer,liver cancer, gallbladder cancer, or colon cancer.

The invention also provides methods for treating lipid metabolismdisorders, biliary disorders, inflammatory disorders, lung disorders,cancer, cardiac disorders including cardiovascular disorders, eyedisorders, oral disorders, blood disorders, liver disorders, skindisorders, prostate disorders, endocrine disorders, and obesity.

Lipid metabolism disorders include, but are not limited to,dyslipidemia, hyperlipidemia, hypercholesterolemia,hypertriglyceridemia, sitosterolemia, familial hypercholesterolemia,xanthoma, combined hyperlipidemia, lecithin cholesterol acyltransferasedeficiency, tangier disease, abetalipoproteinemia, erectile dysfunction,fatty liver disease, and hepatitis.

Billary disorders include gallbladder disorders such as for example,gallstones, gall bladder cancer cholangitis, or primary sclerosingcholangitis; or bile duct disorders such as for example, cholecystitis,bile duct cancer or fascioliasis.

Inflammatory disorders include tissue and organ inflammation such askidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); necrotizingenterocolitis (NEC); pancreatic inflammation (e.g., pancreatis),pancreatic insufficiency, lung inflammation (e.g., bronchitis or asthma)or skin inflammation (e.g., psoriasis, eczema).

Lung Disorders include for example chronic obstructive pulmonary disease(COPD), and fibrosis.

Cancer includes tissue and organ carcinogenesis including metastasessuch as for example gastrointestinal cancer, (e.g., gastric cancer,esophageal cancer, pancreatic cancer colorectal cancer, intestinalcancer, anal cancer, liver cancer, gallbladder cancer, or colon cancer;lung cancer; thyroid cancer; skin cancer (e.g., melanoma); oral cancer;urinary tract cancer (e.g. bladder cancer or kidney cancer); bloodcancer (e.g. myeloma or leukemia) or prostate cancer.

Cardiac disorders include for example, congestive heart failure, tracheacardia hypertension, high cholesterol, or high triglycerides.Cardiovascular disorders include for example aneurysm, angina,atherosclerosis, cerebrovascular accident (stroke),cerebrovasculardisease, congestive heart failure, coronary arterydisease, myocardial infarction (heart attack), or peripheral vasculardisease.

Liver disorders include for example cirrhosis and fibrosis. In addition,GC-C agonist may also be useful to facilitate liver regeneration inliver transplant patients. Eye disorders include for example increasedintra-ocular pressure, glaucoma, dry eyes, retinal degeneration,disorders of tear glands or eye inflammation. Skin disorders include forexample xerosis. Oral disorders include for example dry mouth(xerostomia), Sjögren's syndrome, gum diseases (e.g., periodontaldisease), or salivary gland duct blockage or malfunction. Prostatedisorders include for example benign prostatic hyperplasia (BPH).Endocrine disorders include for example diabetes mellitus,hyperthyroidism, hypothyroidism, and cystic fibrosis.

1.2.1 Therapeutically Effective Dosages

Disorders are treated, prevented or alleviated by administering to asubject, e.g., a mammal such as a human in need thereof, atherapeutically effective dose of a GCC agonist peptide. The presentinvention is based in part on the unexpected results of clinical trialsin humans which demonstrated that the formulations of the invention aretherapeutically effective at much lower doses than predicted based onanimal studies. In accordance with one aspect of the invention, thetherapeutically effective dose is between 0.01 milligrams (mg) and 10 mgper unit dose. The term “unit dose” refers to a single drug deliveryentity, e.g., a tablet, capsule, solution, inhalation, controlledrelease or extended release formulation (e.g. MMX® technology of CosmoPharmaceuticals). In one embodiment, the effective dose is between 0.01mg and 9 mg. In another embodiment, the effective dose is between 0.01mg and 5 mg. In another embodiment, the effective dose is between 0.01mg and 3 mg. In another embodiment, the effective dose is between 0.10mg and 5 mg. In another embodiment, the effective dose is between 0.10mg and 3 mg. In one embodiment, the unit dose is 0.01 mg, 0.05 mg, 0.1mg, 0.2 mg, 0.3 mg, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 5mg, or 10 mg. In one embodiment, the unit dose is 0.3 mg, 1.0 mg, 3.0mg, 9.0 mg, or 9.5 mg.

The GCC agonist peptides may be in a pharmaceutical composition in unitdose form, together with one or more pharmaceutically acceptableexcipients. The amount of peptide present should be sufficient to have apositive therapeutic effect when administered to a patient. Whatconstitutes a “positive therapeutic effect” will depend upon theparticular condition being treated and will include any significantimprovement in a condition readily recognized by one of skill in theart.

The GCC agonists for use in the methods described above are preferablyadministered orally. Dosage forms include solutions, suspensions,emulsions, tablets, and capsules.

The total daily dose can be administered to the patient in a singledose, or in multiple sub-doses. Typically, sub-doses can be administeredtwo to six times per day, preferably two to four times per day, and evenmore preferably two to three times per day. Preferably, a single dailydose is administered.

The GCC agonists may be administered as either the sole active agent orin combination with one or more additional active agents. In all cases,additional active agents should be administered at a dosage that istherapeutically effective using the existing art as a guide. The GCCagonists may be administered in a single composition or sequentiallywith the one or more additional active agents. In one embodiment, theGCC agonist is administered in combination with one or more inhibitorsof cGMP dependent phosphodiesterase such as suldinac sulfone, zaprinast,motapizone, vardenafil, or sildenifil. In another embodiment, the GCCagonist is administered in combination with one or more chemotherapeuticagents. In another embodiment, the GCC agonist is administered incombination with one or more or anti-inflammatory drugs such as steroidsor non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin.

Combination therapy can be achieved by administering two or more agents,e.g., a GCC agonist peptide described herein and another compound, eachof which is formulated and administered separately, or by administeringtwo or more agents in a single formulation. Other combinations are alsoencompassed by combination therapy. For example, two agents can beformulated together and administered in conjunction with a separateformulation containing a third agent. While the two or more agents inthe combination therapy can be administered simultaneously, they neednot be. For example, administration of a first agent (or combination ofagents) can precede administration of a second agent (or combination ofagents) by minutes, hours, days, or weeks. Thus, the two or more agentscan be administered within minutes of each other or within 1, 2, 3, 6,9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or10 weeks of each other. In some cases even longer intervals arepossible. While in many cases it is desirable that the two or moreagents used in a combination therapy be present in within the patient'sbody at the same time, this need not be so.

The GCC agonist peptides described herein may be combined withphosphodiesterase inhibitors, e.g., sulindae sulfone, Zaprinast,sildenafil, vardenafil or tadalafil to further enhance levels of cGMP inthe target tissues or organs.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

1.2.2 Exemplary Agents for Combination Therapy

The GCC agonist formulations of the invention may be administered aloneor in combination with one or more additional therapeutic agents as partof a therapeutic regimen for the treatment or prevention of agastrointestinal disease or disorder. In some embodiments, the GCCagonist formulation comprises one or more additional therapeutic agents.In other embodiments, the GCC agonist is formulated separately from theone or more additional therapeutic agents. In accordance with thisembodiment, the GCC agonist is administered either simultaneously,sequentially, or at a different time than the one or more additionaltherapeutic agents. In one embodiment, the GCC agonist formulation isadministered in combination with one or more additional therapeuticagents selected from the group consisting of phosphodiesteraseinhibitors, cyclic nucleotides (such as cGMP and cAMP), a laxative (suchas SENNA or METAMUCIL), a stool softner, an anti-tumor necrosis factoralpha therapy for IBD (such as REMICADE, ENBREL, or HUMIRA), andanti-inflammatory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASAderivatives and NSAIDS). In certain embodiments, the GCC agonistformulation is administered in combination with an effective dose of aninhibitor of cGMP-specific phosphodiesterase (cGMP-PDE) eitherconcurrently or sequentially with said GCC agonist. cGMP-PDE inhibitorsinclude, for example, suldinac sulfone, zaprinast, motapizone,vardenifil, and sildenafil. In another embodiment, the GCC agonistformulation is administered in combination with inhibitors of cyclicnucleotide transporters. Further examples of therapeutic agents that maybe administered in combination with the GCC agonist formulations of theinvention are given in the following sections.

1.2.2.1 Agents to Treat Gastrointestinal Cancers

The GCC agonist formulations described herein can be used in combinationwith one or more antitumor agents including but not limited toalkylating agents, epipodophyllotoxins, nitrosoureas, anti-metabolites,vinca alkaloids, anthracycline antibiotics, nitrogen mustard agents, andthe like. Particular antitumor agents include tamoxifen, taxol,etoposide, and 5-fluorouracil. In one embodiment, the GCC agonistformulations are used in combination with an antiviral agent or amonoclonal antibody.

Non-limiting examples of antitumor agents that can be used incombination with the GCC agonist formulations of the invention for thetreatment of colon cancer include anti-proliferative agents, agents forDNA modification or repair, DNA synthesis inhibitors, DNA/RNAtranscription regulators, RNA processing inhibitors, agents that affectprotein expression, synthesis and stability, agents that affect proteinlocalization or their ability to exert their physiological action,agents that interfere with protein-protein or protein-nucleic acidinteractions, agents that act by RNA interference, receptor bindingmolecules of any chemical nature (including small molecules andantibodies), targeted toxins, enzyme activators, enzyme inhibitors, generegulators, HSP-90 inhibitors, molecules interfering with microtubulesor other cytoskeletal components or cell adhesion and motility, agentsfor phototherapy, and therapy adjuncts.

Representative anti-proliferative agents include N-acetyl-D-sphingosine(C.sub.2 ceramide), apigenin, berberine chloride,dichloromethylenediphosphonic acid disodium salt, loe-emodine, emodin,HA 14-1, N-hexanoyl-D-sphingosine (C.sub.6 ceramide),7b-hydroxycholesterol, 25-hydroxycholesterol, hyperforin, parthenolide,and rapamycin.

Representative agents for DNA modification and repair includeaphidicolin, bleomycin sulfate, carboplatin, carmustine, chlorambucil,cyclophosphamide monohydrate, cyclophosphamide monohydrate ISOPAC®,cis-diammineplatinum(II) dichloride (Cisplatin), esculetin, melphalan,methoxyamine hydrochloride, mitomycin C, mitoxantrone dihydrochloride,oxaliplatin, and streptozocin.

Representative DNA synthesis inhibitors include (.+−.)amethopterin(methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin,cytosine b-D-arabinofurdnoside (Ara-C), cytosine b-D-arabinofuranoside(Ara-C) hydrochloride, 2-fluoroadenine-9-b-D-arabinofuranoside(Fludarabine des-phosphate; F-ara-A), 5-fluoro-5′-deoxyuridinc,5-fluorouracil, ganciclovir, hydroxyurea, 6-mercaptopurine, and6-thioguanine.

Representative DNA/RNA transcription regulators include actinomycin D,daunorubicin hydrochloride, 5,6-dichlorobenzimidazole1-b-D-ribofuranoside, doxorubicin hydrochloride, homoharringtonine, andidarubicin hydrochloride.

Representative enzyme activators and inhibitors include forskolin,DL-aminoglutethimide, apicidin, Bowman-Birk Inhibitor, butein,(S)-(+)-camptothecin, curcumin, (−)-deguelin, (−)-depudecin, doxycyclinehyclate, etoposide, formestane, fostriecin sodium salt, hispidin,2-imino-1-imidazolidineacetic acid (Cyclocreatine), oxamflatin,4-phenylbutyric acid, roscovitine, sodium valproate, trichostatin A,tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibitor fragment,valproic acid (2-propylpentanoic acid), and XK469.

Representative gene regulators include 5-aza-2′-deoxycytidine,5-azacytidine, cholecalciferol (Vitamin D3), ciglitizone, cyproteroneacetate, 15-deoxy-D.sup.12,14-prostaglandin J.sub.2, epitestosterone,flutamide, glycyrrhizic acid ammonium salt (glycyrrhizin),4-hydroxytamoxifen, mifepristone, procainamide hydrochloride, raloxifenehydrochloride, all trans-retinal (vitamin A aldehyde), retinoic acid(vitamin A acid), 9-cis-retinoic acid, 13-cis-retinoic acid, retinoicacid p-hydroxyanilide, retinol (Vitamin A), tamoxifen, tamoxifen citratesalt, tetradecylthioacetic acid, and troglitazone.

Representative HSP-90 inhibitors include17-(allylamino)-17-demethoxygeldanamycin and geldanamycin.

Representative microtubule inhibitors include colchicines, dolastatin15, nocodazole, taxanes and in particular paclitaxel, podophyllotoxin,rhizoxin, vinblastine sulfate salt, vincristine sulfate salt, andvindesine sulfate salt and vinorelbine (Navelbine) ditartrate salt.

Representative agents for performing phototherapy include photoactiveporphyrin rings, hypericin, 5-methoxypsoralen, 8-methoxypsoralen,psoralen and ursodeoxycholic acid.

Representative agents used as therapy adjuncts include amifostine,4-amino-1,8-naphthalimide, brefeldin A, cimetidine, phosphomycindisodium salt, leuprolide (leuprorelin) acetate salt, luteinizinghormone-releasing hormone (LH-RH) acetate salt, lectin, papaverinehydrochloride, pifithrin-a, (−)-scopolamine hydrobromide, andthapsigargin.

The agents can also be anti-VEGF (vascular endothelial growth factor)agents, as such are known in the art. Several antibodies and smallmolecules are currently in clinical trials or have been approved thatfunction by inhibiting VEGF, such as Avastin (Bevacizumab), SU5416,SU11248 and BAY 43-9006. The agents can also be directed against growthfactor receptors such as those of the EGF/Erb-B family such as EGFReceptor (Iressa or Gefitinib, and Tarceva or Erlotinib), Erb-B2,receptor (Herceptin or Trastuzumab), other receptors (such as Rituximabor Rituxan/MabThera), tyrosine kinases, non-receptor tyrosine kinases,cellular serine/threonine kinases (including MAP kinases), and variousother proteins whose deregulation contribute to oncogenesis (such assmall/Ras family and large/heterotrimeric G proteins). Severalantibodies and small molecules targeting those molecules are currentlyat various stages of development (including approved for treatment or inclinical trials).

In a preferred embodiment, the invention provides a method for treatingcolon cancer in a subject in need thereof by administering to thesubject a GCC agonist formulation in combination with one or moreantitumor agent selected from the group consisting of paclitaxel,docetaxel, tamoxifen, vinorelbine, gemcitabine, cisplatin, etoposide,topotecan, irinotecan, anastrozole, rituximab, trastuzumab, fludarabine,cyclophosphamide, gentuzumab, carboplatin, interferons, and doxorubicin.In a particular embodiment the antitumor agent is paclitaxel. In afurther embodiment, the method further comprises an antitumor agentselected from the group consisting of 5-FU, doxorubicin, vinorelbine,cytoxan, and cisplatin.

1.2.2.2 Agents that Treat Crohn's Disease

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of Crohn's disease.Non-limiting examples of the one or more additional therapeutic agentsinclude sulfasalazine and other mesalamine-containing drugs, generallyknown as 5-ASA agents, such as Asacol, Dipentum, or Pentasa, orinfliximab (REMICADE). In certain embodiments, the one or moreadditional agents is a corticosteroid or an immunosuppressive agent suchas 6-mercaptopurine or azathioprine. In another embodiment, the one ormore additional agents is an antidiarrheal agent such as diphenoxylate,loperamide, or codeine.

1.2.2.3 Agents that Treat Ulcerative Colitis

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of ulcerative colitis.The agents that are used to treat ulcerative colitis overlap with thoseused to treat Chrohn's Disease. Non-limiting examples of the one or moreadditional therapeutic agents that can be used in combination with a GCCagonist formulation of the invention include aminosalicylates (drugsthat contain 5-aminosalicyclic acid (5-ASA)) such as sulfasalazine,olsalazine, mesalamine, and balsalazide. Other therapeutic agents thatcan be used include corticosteroids, such as prednisone andhydrocortisone, immunomodulators, such as azathioprine,6-mercapto-purine (6-MP), cytokines, interleukins, and lymphokines, andanti-TNF-alpha agents, including the thiazolidinediones or glitazonessuch as rosiglitazone and pioglitazone. In one emobidment, the one ormore additional therapeutic agents includes both cyclosporine A and 6-MPor azathioprine for the treatment of active, severe ulcerative colitis.

1.2.2.4 Agents that Treat Constipation/Irritable Bowel Syndrome

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of constipation, such asthat associated with irritable bowel syndrome. Non-limiting examples ofthe one or more additional therapeutic agents include laxatives such asSENNA, MIRALAX, LACTULOSE, PEG, or calcium polycarbophil), stoolsofteners (such as mineral oil or COLACE), bulking agents (such asMETAMUCIL or bran), agents such as ZELNORM (also called tegaserod), andanticholinergic medications such as BENTYL and LEVSIN.

1.2.2.5 Agents for the Treatment of Postoperative Ileus

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of postoperative ileus.Non-limiting examples of the one or more additional therapeutic agentsinclude ENTEREG (alvimopan; formerly called ado lor/ADL 8-2698),conivaptan, and related agents describes in U.S. Pat. No. 6,645,959.

1.2.2.6 Anti-Obesity Agents

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of obesity. Non-limitingexamples of the one or more additional therapeutic agents include 1 lβHSD-I (11-beta hydroxy steroid dehydrogenase type 1) inhibitors, such asBVT 3498, BVT 2733,3-(1-adamantyl)-4-ethyl-5-(ethylthio)-4H-1,2,4-triazole,3-(1-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole,3-adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-1,2,4-triazolo[4,3-a][11]annulene,and those compounds disclosed in WO01/90091, WOO 1/90090, WOO 1/90092and WO02/072084; 5HT antagonists such as those in WO03/037871,WO03/037887, and the like; 5HTIa modulators such as carbidopa,benserazide and those disclosed in U.S. Pat. No. 6,207,699, WO03/031439,and the like; 5HT2c (serotonin receptor 2c) agonists, such as BVT933,DPCA37215, IK264, PNU 22394, WAY161503, R-1065, SB 243213 (Glaxo SmithKline) and YM 348 and those disclosed in U.S. Pat. No. 3,914,250,WO00/77010, WO02/36596, WO02/48124, WO02/10169, WO01/66548, WO02/44152,WO02/51844, WO02/40456, and WO02/40457; 5HT6 receptor modulators, suchas those in WO03/030901, WO03/035061, WO03/039547, and the like;acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa, M.et al, Obesity Research, 9:202-9 (2001) and Japanese Patent ApplicationNo. JP 2000256190; anorectic bicyclic compounds such as 1426 (Aventis)and 1954 (Aventis), and the compounds disclosed in WO00/18749,WO01/32638, WO01/62746, WO01/62747, and WO03/015769; CB 1 (cannabinoid-1receptor) antagonist/inverse agonists such as rimonabant (Acomplia;Sanofi), SR-147778 (Sanofi), SR-141716 (Sanofi), BAY 65-2520 (Bayer),and SLV 319 (Solvay), and those disclosed in patent publications U.S.Pat. Nos. 4,973,587, 5,013,837, 5,081,122, 5,112,820, 5,292,736,5,532,237, 5,624,941, 6,028,084, 6,509,367, 6,509,367, WO96/33159,WO97/29079, WO98/31227, WO98/33765, WO98/37061, WO98/41519, WO98/43635,WO98/43636, WO99/02499, WO00/10967, WO00/10968, WO01/09120, WO01/58869,WO01/64632, WO01/64633, WO01/64634, WO01/70700, WO01/96330, WO02/076949,WO03/006007, WO03/007887, WO03/020217, WO03/026647, WO03/026648,WO03/027069, WO03/027076, WO03/027114, WO03/037332, WO03/040107,WO03/086940, WO03/084943 and EP658546; CCK-A (cholecystokinin-A)agonists, such as AR-R 15849, GI 181771 (GSK), JMV-180, A-71378, A-71623and SR146131 (Sanofi), and those described in U.S. Pat. No. 5,739,106;CNTF (Ciliary neurotrophic factors), such as GI-181771(Glaxo-SmithKline), SR1 46131 (Sanofi Synthelabo), butabindide, PD170,292, and PD 149164 (Pfizer); CNTF derivatives, such as Axokine®(Regeneron), and those disclosed in WO94/09134, WO98/22128, andWO99/43813; dipeptidyl peptidase IV (DP-IV) inhibitors, such asisoleucine thiazolidide, valine pyrrolidide, NVP-DPP728, LAF237, P93/01,P 3298, TSL 225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid; disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)1537-1540), TMC-2A/2B/2C, CD26 inhibtors, FE 999011, P9310/K364, VIP0177, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides asdisclosed by Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22,pp 1163-1166 and 2745-2748 (1996) and the compounds disclosed patentpublications. WO99/38501, WO99/46272, WO99/67279 (Probiodrug),WO99/67278 (Probiodrug), WO99/61431 (Probiodrug), WO02/083128,WO02/062764, WO03/000180, WO03/000181, WO03/000250, WO03/002530,WO03/002531, WO03/002553, WO03/002593, WO03/004498, WO03/004496,WO03/017936, WO03/024942, WO03/024965, WO03/033524, WO03/037327 andEP1258476; growth hormone secretagogue receptor agonists/antagonists,such as NN703, hexarelin, MK-0677 (Merck), SM-130686, CP-424391(Pfizer), LY 444,711 (Eli Lilly), L-692,429 and L-163,255, and such asthose disclosed in U.S. Ser. No. 09/662,448, U.S. provisionalapplication 60/203,335, U.S. Pat. No. 6,358,951, US2002049196,US2002/022637, WO01/56592 and WO02/32888; H3 (histamine H3)antagonist/inverse agonists, such as thioperamide,3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit,iodophenpropit, imoproxifan, GT2394 (Gliatech), and A331440,O-[3-(1H-imidazol-4-yl)propanol]carbamates (Kiec-Kononowicz, K. et al.,Pharmazie, 55:349-55 (2000)), piperidine-containing histamineH3-receptor antagonists (Lazewska, D. et al., Pharmazie, 56:927-32(2001), benzophenone derivatives and related compounds (Sasse, A. etal., Arch. Pharm. (Weinheim) 334:45-52 (2001)), substitutedN-phenylcarbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)),and proxifan derivatives (Sasse, A. et al., J. Med. Chem. 43:3335-43(2000)) and histamine H3 receptor modulators such as those disclosed inWO02/15905, WO03/024928 and WO03/024929; leptin derivatives, such asthose disclosed in U.S. Pat. Nos. 5,552,524, 5,552,523, 5,552,522,5,521,283, WO96/23513, WO96/23514, WO96/23515, WO96/23516, WO96/23517,WO96/23518, WO96/23519, and WO96/23520; leptin, including recombinanthuman leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl humanleptin (Amgen); lipase inhibitors, such as tetrahydrolipstatin(orlistat/Xenical®), Triton WRl 339, RHC80267, lipstatin, teasaponin,diethylumbelliferyl phosphate, FL-386, WAY-121898, Bay-N-3176,valilactone, esteracin, ebelactone A, ebelactone B, and RHC 80267, andthose disclosed in patent publications WO01/77094, U.S. Pat. Nos.4,598,089, 4,452,813, USUS5512565, U.S. Pat. Nos. 5,391,571, 5,602,151,4,405,644, 4,189,438, and 4,242,453; lipid metabolism modulators such asmaslinic acid, erythrodiol, ursolic acid uvaol, betulinic acid, betulin,and the like and compounds disclosed in WO03/011267; Mc4r (melanocortin4 receptor) agonists, such as CHIR86036 (Chiron), ME-10142, ME-10145,and HS-131 (Melacure), and those disclosed in PCT publication Nos.WO99/64002, WO00/74679, WOO 1/991752, WOO 1/25192, WOO 1/52880, WOO1/74844, WOO 1/70708, WO01/70337, WO01/91752, WO02/059095, WO02/059107,WO02/059108, WO02/059117, WO02/06276, WO02/12166, WO02/11715,WO02/12178, WO02/15909, WO02/38544, WO02/068387, WO02/068388,WO02/067869, WO02/081430, WO03/06604, WO03/007949, WO03/009847,WO03/009850, WO03/013509, and WO03/031410; Mc5r (melanocortin 5receptor) modulators, such as those disclosed in WO97/19952, WO00/15826,WO00/15790, US20030092041; melanin-concentrating hormone 1 receptor(MCHR) antagonists, such as T-226296 (Takeda), SB 568849, SNP-7941(Synaptic), and those disclosed in patent publications WOO 1/21169,WO01/82925, WO01/87834, WO02/051809, WO02/06245, WO02/076929,WO02/076947, WO02/04433, WO02/51809, WO02/083134, WO02/094799,WO03/004027, WO03/13574, WO03/15769, WO03/028641, WO03/035624,WO03/033476, WO03/033480, JP13226269, and JP1437059; mGluR5 modulatorssuch as those disclosed in WO03/029210, WO03/047581, WO03/048137,WO03/051315, WO03/051833, WO03/053922, WO03/059904, and the like;serotoninergic agents, such as fenfluramine (such as Pondimin®(Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)-,hydrochloride), Robbins), dexfenfluramine (such as Redux®(Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)-,hydrochloride), Interneuron) and sibutramine ((Meridia®,Knoll/Reductil™) including racemic mixtures, as optically pure isomers(+) and (−), and pharmaceutically acceptable salts, solvents, hydrates,clathrates and prodrugs thereof including sibutramine hydrochloridemonohydrate salts thereof, and those compounds disclosed in U.S. Pat.Nos. 4,746,680, 4,806,570, and 5,436,272, US20020006964, WOO 1/27068,and WOO 1/62341; NE (norepinephrine) transport inhibitors, such as GW320659, despiramine, talsupram, and nomifensine; NPY 1 antagonists, suchas BIBP3226, J-115814, MO 3304, LY-357897, CP-671906, GI-264879A, andthose disclosed in U.S. Pat. No. 6,001,836, WO96/14307, WO01/23387,WO99/51600, WO01/85690, WO01/85098, WO01/85173, and WO01/89528; NPY5(neuropeptide Y Y5) antagonists, such as 152,804, GW-569180A,GW-594884A, GW-587081X, GW-548118X, FR235208, FR226928, FR240662,FR252384, 1229U91, GI-264879A, CGP71683A, LY-377897, LY-366377,PD-160170, SR-120562A, SR-120819A, JCF-104, and H409/22 and thosecompounds disclosed in patent publications U.S. Pat. Nos. 6,140,354,6,191,160, 6,218,408, 6,258,837, 6,313,298, 6,326,375, 6,329,395,6,335,345, 6,337,332, 6,329,395, 6,340,683, EP01010691, EP-01044970,WO97/19682, WO97/20820, WO97/20821, WO97/20822, WO97/20823, WO98/27063,WO00/107409, WO00/185714, WO00/185730, WO00/64880, WO00/68197,WO00/69849, WO/0113917, WO01/09120, WO01/14376, WO01/85714, WO01/85730,WO01/07409, WO01/02379, WO01/23388, WO01/23389, WOO 1/44201, WO01/62737,WO01/62738, WO01/09120, WO02/20488, WO02/22592, WO02/48152, WO02/49648,WO02/051806, WO02/094789, WO03/009845, WO03/014083, WO03/022849,WO03/028726 and Norman et al, J. Med. Chem. 43:4288-4312 (2000); opioidantagonists, such as nalmefene (REVEX®), 3-methoxynaltrexone,methylnaltrexone, naloxone, and naltrexone (e.g. PT901; PainTherapeutics, Inc.) and those disclosed in US20050004155 and WO00/21509;orexin antagonists, such as SB-334867-A and those disclosed in patentpublications WO01/96302, WO01/68609, WO02/44172, WO02/51232, WO02/51838,WO02/089800, WO02/090355, WO03/023561, WO03/032991, and WO03/037847; PDEinhibitors (e.g. compounds which slow the degradation of cyclic AMP(cAMP) and/or cyclic GMP (cGMP) by inhibition of the phosphodiesterases,which can lead to a relative increase in the intracellular concentrationof cAMP and cGMP; possible PDE inhibitors are primarily those substanceswhich are to be numbered among the class consisting of the PDE3inhibitors, the class consisting of the PDE4 inhibitors and/or the classconsisting of the PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors) such as those disclosed in patent publicationsDE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801,DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481,DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948,EP0075436, EP0096517, EPOl 12987, EPOl 16948, EP0150937, EP0158380,EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725,EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811,EP0470805, EP0482208, EP0490823, EP0506194, EP0511865, EP0527117,EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479,JP92234389, JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931,WO9117991, WO9200968, WO9212961, WO9307146, WO9315044, WO9315045,WO9318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751,WO9325517, WO9402465, WO9406423, WO9412461, WO9420455, WO9422852,WO9425437, WO9427947, WO9500516, WO9501980, WO9503794, WO9504045,WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627,WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399,WO9519362, WO9522520, WO9524381, WO9527692, WO9528926, WO9535281,WO9535282, WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DEl116676, DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345U.S. Pat. No. 6,331,543, US20050004222 (including those disclosed informulas I-XIII and paragraphs 37-39, 85-0545 and 557-577), WO9307124,EP0163965, EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399, aswell as PDE5 inhibitors (such as RX-RA-69, SCH-51866, KT-734,vesnarinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702 andsildenafil (Viagra™)), PDE4 inhibitors (such as etazolate, ICI63197,RP73401, imazolidinone (RO-20-1724), MEM 1414 (R1533/R1500; PharmaciaRoche), denbufylline, rolipram, oxagrelate, nitraquazone, Y-590,DH-6471, SKF-94120, motapizone, lixazinone, indolidan, olprinone,atizoram, KS-506-G, dipamfylline, BMY-43351, atizoram, arofylline,filaminast, PDB-093, UCB-29646, CDP-840, SKF-107806, piclamilast,RS-17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572,SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, mopidamol,anagrelide, ibudilast, amrinone, pimobendan, cilostazol, quazinone andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide,PDE3 inhibitors (such as ICI153, 100, bemorandane (RWJ 22867), MCI-154,UD-CG 212, sulmazole, ampizone, cilostamide, carbazeran, piroximone,imazodan, CI-930, siguazodan, adibendan, saterinone, SKF-95654,SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP-307,revizinone, NM-702, WIN-62582 and WIN-63291, enoximone and milrinone,PDE3/4 inhibitors (such as benafentrine, trequinsin, ORG-30029,zardaverine, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andtolafentrine) and other PDE inhibitors (such as vinpocetin, papaverine,enprofylline, cilomilast, fenoximone, pentoxifylline, roflumilast,tadalafil (Cialis®), theophylline, and vardenafil (Levitra®);Neuropeptide Y2 (NPY2) agonists include but are not limited to:polypeptide YY and fragments and variants thereof (e.g. YY3-36 (PYY3-36)(N. Engl. J. Med. 349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY(SEQ ID NO:XXX)) and PYY agonists such as those disclosed in WO02/47712,WO03/026591, WO03/057235, and WO03/027637; serotonin reuptakeinhibitors, such as, paroxetine, fluoxetine (Prozac™), fluvoxamine,sertraline, citalopram, and imipramine, and those disclosed in U.S. Pat.Nos. 6,162,805, 6,365,633, WO03/00663, WOO 1/27060, and WOO 1/162341;thyroid hormone β agonists, such as KB-2611 (KaroBioBMS), and thosedisclosed in WO02/15845, WO97/21993, WO99/00353, GB98/284425, U.S.Provisional Application No. 60/183,223, and Japanese Patent ApplicationNo. JP 2000256190; UCP-I (uncoupling protein-1), 2, or 3 activators,such as phytanic acid,4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoicacid (TTNPB), retinoic acid, and those disclosed in WO99/00123; β3 (betaadrenergic receptor 3) agonists, such as AJ9677/TAK677(Dainippon/Takeda), L750355 (Merck), CP331648 (Pfizer), CL-316,243, SB418790, BRL-37344, L-796568, BMS-196085, BRL-35135A, CGP12177A, BTA-243,GW 427353, Trecadrine, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604(Lilly), SR 59119A, and those disclosed in U.S. Pat. Nos. 5,541,204,5,770,615, 5,491,134, 5,776,983, US488064, U.S. Pat. Nos. 5,705,515,5,451,677, WO94/18161, WO95/29159, WO97/46556, WO98/04526 andWO98/32753, WO01/74782, WO02/32897, WO03/014113, WO03/016276,WO03/016307, WO03/024948, WO03/024953 and WO03/037881; noradrenergicagents including, but not limited to, diethylpropion (such as Tenuate®(1-propanone, 2-(diethylamino)-1-phenyl-, hydrochloride), Merrell),dextroamphetamine (also known as dextroamphetamine sulfate,dexamphetamine, dexedrine, Dexampex, Ferndex, Oxydess II, Robese,Spancap #1), mazindol ((or5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-01) such asSanorex®, Novartis or Mazanor®, Wyeth Ayerst), phenylpropanolamine (orBenzenemethanol, alpha-(1-aminoethyl)-, hydrochloride), phentermine ((orPhenol, 3-[[4,5-duhydro-1H-imidazol-2-yl)ethyl](4-methylpheny-1)amino],monohydrochloride) such as Adipex-P®, Lemmon, FASTIN®, Smith-KlineBeecham and Ionamin®, Medeva), phendimetrazine ((or(2S,3S)-3,4-Dimethyl-2phenylmorpholine L-(+)-tartrate (1:1)) such asMetra® (Forest), Plegine® (Wyeth-Ayerst), Prelu-2® (BoehringerIngelheim), and Statobex® (Lemmon), phendamine tartrate (such asThephorin®(2,3,4,9-Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridineL-(+)-tartrate (1:1)), Hoffmann-LaRoche), methamphetamine (such asDesoxyn®, Abbot ((S)—N, (alpha)-dimethylbenzeneethanaminehydrochloride)), and phendimetrazine tartrate (such as Bontril®Slow-Release Capsules, Amarin (-3,4-Dimethyl-2-phenylmorpholineTartrate); fatty acid oxidation upregulator/inducers such as Famoxin®(Genset); monamine oxidase inhibitors including but not limited tobefloxatone, moclobemide, brofaromine, phenoxathine, esuprone, befol,toloxatone, pirlindol, amiflamine, sercloremine, bazinaprine,lazabemide, milacemide, caroxazone and other certain compounds asdisclosed by WO01/12176; and other anti-obesity agents such as 5HT-2agonists, ACC (acetyl-CoA carboxylase) inhibitors such as thosedescribed in WO03/072197, alpha-lipoic acid (alpha-LA), AOD9604,appetite suppressants such as those in WO03/40107, ATL-962 (AlizymePLC), benzocaine, benzphetamine hydrochloride (Didrex), bladderwrack(focus vesiculosus), BRS3 (bombesin receptor subtype 3) agonists,bupropion, caffeine, CCK agonists, chitosan, chromium, conjugatedlinoleic acid, corticotropin-releasing hormone agonists,dehydroepiandrosterone, DGAT1 (diacylglycerol acyltransferase 1)inhibitors, DGAT2 (diacylglycerol acyltransferase 2) inhibitors,dicarboxylate transporter inhibitors, ephedra, exendin-4 (an inhibitorof glp-1) FAS (fatty acid synthase) inhibitors (such as Cerulenin andC75), fat resorption inhibitors (such as those in WO03/053451, and thelike), fatty acid transporter inhibitors, natural water soluble fibers(such as psyllium, plantago, guar, oat, pectin), galanin antagonists,galega (Goat's Rue, French Lilac), garcinia cambogia, germander(teucrium chamaedrys), ghrelin antibodies and ghrelin antagonists (suchas those disclosed in WO01/87335, and WO02/08250), polypeptide hormonesand variants thereof which affect the islet cell secretion, such as thehormones of the secretin/gastric inhibitory polypeptide (GIP)/vasoactiveintestinal polypeptide (VIP)/pituitary adenylate cyclase activatingpolypeptide (PACAP)/glucagon-like polypeptide II(GLP-II)/glicentin/glucagon gene family and/or those of theadrenomedullin/amylin/calcitonin gene related polypeptide (CGRP) genefamily includingGLP-1 (glucagon-like polypeptide 1) agonists (e.g. (1)exendin-4, (2) those GLP-I molecules described in US20050130891including GLP-1(7-34), GLP-1(7-35), GLP-1(7-36) or GLP-1(7-37) in itsC-terminally carboxylated or amidated form or as modified GLP-Ipolypeptides and modifications thereof including those described inparagraphs 17-44 of US20050130891, and derivatives derived fromGLP-1-(7-34)COOH and the corresponding acid amide are employed whichhave the following general formula:R—NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH₂ wherein R═H or an organiccompound having from 1 to 10 carbon atoms. Preferably, R is the residueof a carboxylic acid. Particularly preferred are the followingcarboxylic acid residues: formyl, acetyl, propionyl, isopropionyl,methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl.) andglp-1 (glucagon-like polypeptide-1), glucocorticoid antagonists, glucosetransporter inhibitors, growth hormone secretagogues (such as thosedisclosed and specifically described in U.S. Pat. No. 5,536,716),interleukin-6 (IL-6) and modulators thereof (as in WO03/057237, and thelike), L-carnitine, Mc3r (melanocortin 3 receptor) agonists, MCH2R(melanin concentrating hormone 2R) agonist/antagonists, melaninconcentrating hormone antagonists, melanocortin agonists (such asMelanotan II or those described in WO 99/64002 and WO 00/74679), nomameherba, phosphate transporter inhibitors, phytopharm compound 57 (CP644,673), pyruvate, SCD-I (stearoyl-CoA desaturase-1) inhibitors, T71(Tularik, Inc., Boulder Colo.), Topiramate (Topimax®, indicated as ananti-convulsant which has been shown to increase weight loss),transcription factor modulators (such as those disclosed inWO03/026576), β-hydroxy steroid dehydrogenase-1 inhibitors (β-HSD-I),β-hydroxy-β-methylbutyrate, p57 (Pfizer), Zonisamide (Zonegran™indicated as an anti-epileptic which has been shown to lead to weightloss), and the agents disclosed in US20030119428 paragraphs 20-26.

1.2.2.7 Phosphodiesterase inhibitors

In certain embodiments, the regimen of combination therapy includes theadministration of one or more phosphodiesterase (“PDE”) inhibitors. PDEinhibitors slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP(cGMP) by inhibiting phosphodiesterases, which can lead to a relativeincrease in the intracellular concentration of cAMP and/or cGMP.Non-limiting examples of PDE inhibitors that can be used in combinationwith the GCC agonists of the invention include PDE3 inhibitors, PDE4inhibitors and/or PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors. Non-limiting examples of such PDE inhibitors aredescribed in the following patent applications and patents: DE1470341,DE2108438, DE2123328, DE2305339, DE2305575, DE2315801, DE2402908,DE2413935, DE2451417, DE2459090, DE2646469, DE2727481, DE2825048,DE2837161, DE2845220, DE2847621, DE2934747, DE3021792, DE3038166,DE3044568, EP000718, EP0008408, EP0010759, EP0059948, EP0075436,EP0096517, EPOl 12987, EPOl 16948, EP0150937, EP0158380, EP0161632,EP0161918, EP0167121, EP0199127, EP0220044, EP0247725, EP0258191,EP0272910, EP0272914, EP0294647, EP0300726, EP0335386, EP0357788,EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805,EP0482208, EP0490823, EP0506194, EP0511865, EP0527117, EP0626939,EP0664289, EP0671389, EP0685474, EP0685475, EP0685479, JP92234389,JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931, WO9117991,WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024,WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517,WO9402465, WO9406423, WO9412461, WO9420455, WO9422852, WO9425437,WO9427947, WO9500516, WO9501980, WO9503794, WO9504045, WO9504046,WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836,WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362,WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282,WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DEl 116676,DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345 U.S.Pat. No. 6,331,543, US20050004222 (including those disclosed in formulasI—XIII and paragraphs 37-39, 85-0545 and 557-577) and WO9307124,EP0163965, EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399.PDE5 inhibitors which may be mentioned by way of example are RX-RA-69,SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355,BF/GP-385, NM-702 and sildenafil (Viagra®). PDE4 inhibitors which may bementioned by way of example are RO-20-1724, MEM 1414 (R1533/R1500;Pharmacia Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE,Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZINONE, INDOLIDAN, OLPRINONE,ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, AROFYLLINE,FILAMINAST, PDB-093, UCB-29646, CDP-840, SKF-107806, PICLAMILAST,RS-17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572,SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL,ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL, QUAZINONE andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide.PDE3 inhibitors which may be mentioned by way of example are SULMAZOLE,AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930,SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85,EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702,WIN-62582 and WIN-63291, ENOXIMONE and MILRINONE. PDE3/4 inhibitorswhich may be mentioned by way of example are BENAFENTRINE, TREQUINSIN,ORG-30029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andTOLAFENTRINE. Other PDE inhibitors include: cilomilast, pentoxifylline,roflumilast, tadalafil (Cialis®), theophylline, and vardenafil(Levitra®), zaprinast (PDE5 specific). GCC AGONIST

1.2.2.8 Analgesic Agents

In certain embodiments, the regimen of combination therapy includes theadministration of one or more analgesic agents, e.g., an analgesiccompound or an analgesic polypeptide. In some embodiments, the GCCagonist formulation is administered simultaneously or sequentially withone or more analgesic agents. In other embodiments, the GCC agonist iscovalently linked or attached to an analgesic agent to create atherapeutic conjugate. Non-limiting examples of analgesic agents thatcan be used include calcium channel blockers, 5HT receptor antagonists(for example 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptoragonists (loperamide, fedotozine, and fentanyl), NK1 receptorantagonists, CCK receptor agonists (e.g., loxiglumide), NK1 receptorantagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptakeinhibitors (NSRI), vanilloid and cannabanoid receptor agonists, andsialorphin. Further examples of analgesic agents in the various classesare known in the art.

In one embodiment, the analgesic agent is an analgesic polypeptideselected from the group consisting of sialorphin-related polypeptides,including those comprising the amino acid sequence QHNPR (SEQ ID NO:239), including: VQHNPR (SEQ ID NO: 240); VRQHNPR (SEQ ID NO: 241);VRGQHNPR (SEQ ID NO: 242); VRGPQHNPR (SEQ ID NO: 243); VRGPRQHNPR (SEQID NO: 244); VRGPRRQHNPR (SEQ ID NO: 245); and RQHNPR (SEQ ID NO: 246).Sialorphin-related polypeptides bind to neprilysin and inhibitneprilysin-mediated breakdown of substance P and Met-enkephalin. Thus,compounds or polypeptides that are inhibitors of neprilysin are usefulanalgesic agents which can be administered with the GCC agonistsdescribed herein or covalently linked to a GCC agonist to form atherapeutic conjugate. Sialorphin and related polypeptides are describedin U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO 02/051435 A2.

In another embodiment, a GCC agonist formulation of the invention isadministered as part of a regimen of combination therapy with an opioidreceptor antagonist or agonist. In one embodiment, the GCC agonist andthe opioid receptor antagonist or agonist are linked via a covalentbond. Non-limiting examples of opioid receptor antagonists includenaloxone, naltrexone, methyl nalozone, nalmefene, cypridime, betafunaltrexamine, naloxonazine, naltrindole, nor-binaltorphimine,enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine),trimebutine, vasoactive intestinal polypeptide, gastrin, glucagons.Non-limiting examples of opioid receptor agonists include fedotozine,asimadoline, and ketocyclazocine, the compounds described in WO03/097051and WO05/007626, morphine, diphenyloxylate, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH 2; WO 01/019849 A1), and loperamide.

Further non-limiting examples of analgesic agents that can be used in aregimen of combination therapy along with the GCC agonist formulationsof the invention include the dipeptide Tyr-Arg (kyotorphin); thechromogranin-derived polypeptide (CgA 47-66; See, e.g., Ghia et al. 2004Regulatory polypeptides 119:199); CCK receptor agonists such ascaerulein; conotoxin polypeptides; peptide analogs of thymulin (FRApplication 2830451); CCK (CCKa or CCKb) receptor antagonists, includingloxiglumide and dexloxiglumide (the R-isomer of loxiglumide) (WO88/05774); 5-HT4 agonists such as tegaserod (Zelnorm®), mosapride,metoclopramide, zacopride, cisapride, renzapride, benzimidazolonederivatives such as BIMU 1 and BIMU 8, and lirexapride; calcium channelblockers such as ziconotide and related compounds described in, forexample, EP625162B1, U.S. Pat. Nos. 5,364,842, 5,587,454, 5,824,645,5,859,186, 5,994,305, 6,087,091, 6,136,786, WO 93/13128 A1, EP 1336409A1, EP 835126 A1, EP 835126 B1, U.S. Pat. Nos. 5,795,864, 5,891,849,6,054,429, WO 97/01351 A1; NK-I, receptor antagonists such as aprepitant(Merck & Co Inc), vofopitant, ezlopitant (Pfizer, Inc.), R-673(Hoffmann-La Roche Ltd), SR-48968 (Sanofi Synthelabo), CP-122,721(Pfizer, Inc.), GW679769 (Glaxo Smith Kline), TAK-637 (Takeda/Abbot),SR-14033, and related compounds described in, for example, EP 873753 A1,US 20010006972 A1, US 20030109417 A1, WO 01/52844 A1 (for a review seeGiardina et al. 2003. Drugs 6:758); NK-2 receptor antagonists such asnepadutant (Menarini Ricerche SpA), saredutant (Sanoft-Synthelabo),GW597599 (Glaxo Smith Kline), SR-144190 (Sanofi-Synthelabo) andUK-290795 (Pfizer Inc); NK3 receptor antagonists such as osanetant(SR-142801; Sanoft-Synthelabo), SSR-241586, talnetant and relatedcompounds described in, for example, WO 02/094187 A2, EP 876347 A1, WO97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/1 1090, WO 95/28418, WO97/19927, and Boden et al. (J Med Chem. 39:1664-75, 1996);norepinephrine-serotonin reuptake inhibitors (NSRI) such as milnacipranand related compounds described in WO 03/077897; and vanilloid receptorantagonists such as arvanil and related compouds described in WO01/64212 A1.

In addition to sialorphin-related polypeptides, analgesic polypeptidesinclude: AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin,lupron, ziconotide, and substance P.

1.2.2.9 Insulin and Insulin Modulating Agents

The GCC agonist peptides described herein can be used in combinationtherapy with insulin and related compounds including primate, rodent, orrabbit insulin including biologically active variants thereof includingallelic variants, more preferably human insulin available in recombinantform. Sources of human insulin include pharmaceutically acceptable andsterile formulations such as those available from Eli Lilly(Indianapolis, Ind. 46285) as Humulin™ (human insulin rDNA origin). See,the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) MedicalEconomics, Thomson Healthcare (disclosing other suitable humaninsulins).

The GCC peptides described herein can also be used in combinationtherapy with agents that can boost insulin effects or levels of asubject upon administration, e.g. glipizide and/or rosiglitazone. Thepolypeptides and agonists described herein can be used in combitherapywith SYMLIN® (pramlintide acetate) and Exenatide® (synthetic exendin-4;a 39 aa polypeptide).

1.2.2.10 Anti-Hypertensive Agents

The GCC agonist peptides described herein can be used in combinationtherapy with an anti-hypertensive agent including but not limited to:(1) diuretics, such as thiazides, including chlorthalidone,chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide,polythiazide, and hydrochlorothiazide; loop diuretics, such asbumetanide, ethacrynic acid, furosemide, and torsemide; potassiumsparing agents, such as amiloride, and triamterene; carbonic anhydraseinhibitors, osmotics (such as glycerin) and aldosterone antagonists,such as spironolactone, epirenone, and the like; (2) beta-adrenergicblockers such as acebutolol, atenolol, betaxolol, bevantolol,bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esmolol,indenolol, metaprolol, nadolol, nebivolol, penbutolol, pindolol,propanolol, sotalol, tertatolol, tilisolol, and timolol, and the like;(3) calcium channel blockers such as amlodipine, aranidipine,azelnidipine, barnidipine, benidipine, bepridil, cinaldipine,clevidipine, diltiazem, efonidipine, felodipine, gallopamil, isradipine,lacidipine, lemildipine, lercanidipine, nicardipine, nifedipine,nilvadipine, nimodepine, nisoldipine, nitrendipine, manidipine,pranidipine, and verapamil, and the like; (4) angiotensin convertingenzyme (ACE) inhibitors such as benazepril; captopril; ceranapril;cilazapril; delapril; enalapril; enalopril; fosinopril; imidapril;lisinopril; losinopril; moexipril; quinapril; quinaprilat; ramipril;perindopril; perindropril; quanipril; spirapril; tenocapril;trandolapril, and zofenopril, and the like; (5) neutral endopeptidaseinhibitors such as omapatrilat, cadoxatril and ecadotril, fosidotril,sampatrilat, AVE7688, ER4030, and the like; (6) endothelin antagonistssuch as tezosentan, A308165, and YM62899, and the like; (7) vasodilatorssuch as hydralazine, clonidine, minoxidil, and nicotinyl alcohol, andthe like; (8) angiotensin II receptor antagonists such as aprosartan,candesartan, eprosartan, irbesartan, losartan, olmesartan, pratosartan,tasosartan, telmisartan, valsartan, and EXP-3137, FI6828K, and RNH6270,and the like; (9) α/β adrenergic blockers such as nipradilol, arotinololand amosulalol, and the like; (10) alpha 1 blockers, such as terazosin,urapidil, prazosin, tamsulosin, bunazosin, trimazosin, doxazosin,naftopidil, indoramin, WHP 164, and XENOlO, and the like; (11) alpha 2agonists such as lofexidine, tiamenidine, moxonidine, rilmenidine andguanobenz, and the like; (12) aldosterone inhibitors, and the like; and(13) angiopoietin-2-binding agents such as those disclosed inWO03/030833. Specific anti-hypertensive agents that can be used incombination with polypeptides and agonists described herein include, butare not limited to: diuretics, such as thiazides (e.g., chlorthalidone,cyclothiazide (CAS RN 2259-96-3), chlorothiazide (CAS RN 72956-09-3,which may be prepared as disclosed in U.S. Pat. No. 2,809,194),dichlorophenamide, hydroflumethiazide, indapamide, polythiazide,bendroflumethazide, methyclothazide, polythiazide, trichlormethazide,chlorthalidone, indapamide, metolazone, quinethazone, althiazide (CAS RN5588-16-9, which may be prepared as disclosed in British Patent No.902,658), benzthiazide (CAS RN 91-33-8, which may be prepared asdisclosed in U.S. Pat. No. 3,108,097), buthiazide (which may be preparedas disclosed in British Patent Nos. 861,367), and hydrochlorothiazide),loop diuretics (e.g. bumetanide, ethacrynic acid, furosemide, andtorasemide), potassium sparing agents (e.g. amiloride, and triamterene(CAS Number 396-01-O)), and aldosterone antagonists (e.g. spironolactone(CAS Number 52-01-7), epirenone, and the like); β-adrenergic blockerssuch as Amiodarone (Cordarone, Pacerone), bunolol hydrochloride (CAS RN31969-05-8, Parke-Davis), acebutolol (±N-[3-Acetyl-4-[2-hydroxy-3-[(1methylethyl)amino]propoxy]phenyl]-butanamide, or(±)-3′-Acetyl-4′-[2-hydroxy-3-(isopropylamino) propoxy] butyranilide),acebutolol hydrochloride (e.g. Sectral®, Wyeth-Ayerst), alprenololhydrochloride (CAS RN 13707-88-5 see Netherlands Patent Application No.6,605,692), atenolol (e.g. Tenormin®, AstraZeneca), carteololhydrochloride (e.g. Cartrol® Filmtab®, Abbott), Celiprolol hydrochloride(CAS RN 57470-78-7, also see in U.S. Pat. No. 4,034,009), cetamololhydrochloride (CAS RN 77590-95-5, see also U.S. Pat. No. 4,059,622),labetalol hydrochloride (e.g. Normodyne®, Schering), esmololhydrochloride (e.g. Brevibloc®, Baxter), levobetaxolol hydrochloride(e.g. Betaxon™ Ophthalmic Suspension, Alcon), levobunolol hydrochloride(e.g. Betagan® Liquifilm® with C CAP® Compliance Cap, Allergan), nadolol(e.g. Nadolol, Mylan), practolol (CAS RN 6673-35-4, see also U.S. Pat.No. 3,408,387), propranolol hydrochloride (CAS RN 318-98-9), sotalolhydrochloride (e.g. Betapace AF™, Berlex), timolol (2-Propanol,1-[(1,1-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-,hemihydrate, (S)—, CAS RN 91524-16-2), timolol maleate(S)-1-[(1,1-dimethylethyl)amino]-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl] oxy]-2-propanol(Z)-2-butenedioate (1:1) salt, CAS RN 26921-17-5), bisoprolol(2-Propanol,1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-meth-ylethyl)amino]-,(±), CAS RN 66722-44-9), bisoprolol fumarate (such as(±)-1-[4-[[2-(1-Methylethoxy)ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol(E)-2-butenedioate (2:1) (salt), e.g., Zebeta™, Lederle Consumer),nebivalol (2H-1-Benzopyran-2-methanol,αα′-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, CAS RN 99200-09-6see also U.S. Pat. No. 4,654,362), cicloprolol hydrochloride, such2-Propanol,1-[4-[2-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-,hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride(2-Propanol, 1-[1-methylethy)-amino]-3-(1-naphthalenyloxy)-hydrochloride(CAS RN 13071-11-9), diacetolol hydrochloride (Acetamide,N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy] [phenyl]-,monohydrochloride CAS RN 69796-04-9), dilevalol hydrochloride(Benzamide,2-hydroxy-5-[1-hydroxy-2-[1-methyl-3-phenylpropyl)amino]ethyl]-,monohydrochloride, CAS RN 75659-08-4), exaprolol hydrochloride(2-Propanol, 1-(2-cyclohexylphenoxy)-3-[(1-methylethyl)amino]-,hydrochloride CAS RN 59333-90-3), flestolol sulfate (Benzoic acid,2-fluro-,3-[[2-[aminocarbonyl)amino]-dimethylethyl]amino]-2-hydroxypropyl ester,(+)-sulfate (1:1) (salt), CAS RN 88844-73-9; metalol hydrochloride(Methanesulfonamide, N-[4-[1-hydroxy-2-(methyl amino)propyl]phenyl]-,monohydrochloride CAS RN 7701-65-7), metoprolol 2-Propanol,1-[4-(2-methoxyethyl)phenoxy]-3-[1-methylethyl)amino]-; CAS RN37350-58-6), metoprolol tartrate (such as 2-Propanol,1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, e.g.,Lopressor®, Novartis), pamatolol sulfate (Carbamic acid,[2-[4-[2-hydroxy-3-[(1-methylethyl)amino]propoxyl]phenyl]-ethyl]-,methyl ester, (±) sulfate (salt) (2:1), CAS RN 59954-01-7), penbutololsulfate (2-Propanol,1-(2-cyclopentylphenoxy)-3-[1,1-dimethyle-thyl)amino] 1, (S)-, sulfate(2:1) (salt), CAS RN 38363-32-5), practolol (Acetamide,N-[4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, CAS RN6673-35-4) tiprenolol hydrochloride (Propanol,1-[(1-methylethyl)amino]-3-[2-(methylthio)-phenoxy]-, hydrochloride,(±), CAS RN 39832-43-4), tolamolol (Benzamide,4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl] amino] ethoxyl]-, CAS RN38103-61-6), bopindolol, indenolol, pindolol, propanolol, tertatolol,and tilisolol, and the like; calcium channel blockers such as besylatesalt of amlodipine (such as3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylatebenzenesulphonate, e.g., Norvasc®, Pfizer), clentiazem maleate(1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methoxyphenyl)-(2S-cis)-,(Z)-2-butenedioate (1:1), see also U.S. Pat. No. 4,567,195), isradipine(3,5-Pyridinedicarboxylic acid,4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-methylethylester,(±)-4(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate,see also U.S. Pat. No. 4,466,972); nimodipine (such as is isopropyl(2-methoxyethyl) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate,e.g. Nimotop®, Bayer), felodipine (such as ethyl methyl4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate-,e.g. Plendil® Extended-Release, AstraZeneca LP), nilvadipine(3,5-Pyridinedicarboxylic acid,2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-, 3-methyl5-(1-methylethyl) ester, also see U.S. Pat. No. 3,799,934), nifedipine(such as 3, 5-pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester, e.g.,Procardia XL® Extended Release Tablets, Pfizer), diltiazem hydrochloride(such as 1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (±)-cis., e.g., Tiazac®, Forest), verapamilhydrochloride (such as benzeneacetronitrile,(alpha)-[[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Isoptin® SR, Knoll Labs), teludipine hydrochloride(3,5-Pyridinedicarboxylic acid,2-[(dimethylamino)methyl]4-[2-[(1E)-3-(1,1-dimethylethoxy)-3-oxo-1-propenyl]phenyl]-1,4-dihydro-6-methyl-,diethyl ester, monohydrochloride) CAS RN 108700-03-4), belfosdil(Phosphonic acid, [2-(2-phenoxy ethyl)-1,3-propane-diyl]bis-, tetrabutylester CAS RN 103486-79-9), fostedil (Phosphonic acid,[[4-(2-benzothiazolyl)phenyl]methyl]-, diethyl ester CAS RN 75889-62-2),aranidipine, azelnidipine, barnidipine, benidipine, bepridil,cinaldipine, clevidipine, efonidipine, gallopamil, lacidipine,lemildipine, lercanidipine, monatepil maleate (1-Piperazinebutanamide,N-(6, 11-dihydrodibenzo(b,e)thiepin-11-yl)4-(4-fluorophenyl)-, (+)-,(Z)-2-butenedioate (1:1)(±)-N-(6,11-Dihydrodibenzo(b,e)thiep-in-11-yl)-4-(p-fluorophenyl)-1-piperazinebutyramidemaleate (1:1) CAS RN 132046-06-1), nicardipine, nisoldipine,nitrendipine, manidipine, pranidipine, and the like; T-channel calciumantagonists such as mibefradil; angiotensin converting enzyme (ACE)inhibitors such as benazepril, benazepril hydrochloride (such as3-[[1-(ethoxycarbonyl)-3-phenyl-(1 S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3 S)-benzazepine-1-acetic acidmonohydrochloride, e.g., Lotrel®, Novartis), captopril (such as1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, e.g., Captopril, Mylan,CAS RN 62571-86-2 and others disclosed in U.S. Pat. No. 4,046,889),ceranapril (and others disclosed in U.S. Pat. No. 4,452,790), cetapril(alacepril, Dainippon disclosed in Eur. Therap. Res. 39:671 (1986);40:543 (1986)), cilazapril (Hoffman-LaRoche) disclosed in J. Cardiovasc.Pharmacol. 9:39 (1987), indalapril (delapril hydrochloride(2H-1,2,4-Benzothiadiazine-7-sulfonamide,3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide CAS RN2259-96-3); disclosed in U.S. Pat. No. 4,385,051), enalapril (and othersdisclosed in U.S. Pat. No. 4,374,829), enalopril, enaloprilat,fosinopril, ((such as L-proline,4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy) propoxy](4-phenylbutyl)phosphinyl]acetyl]-, sodium salt, e.g., Monopril, Bristol-Myers Squibband others disclosed in U.S. Pat. No. 4,168,267), fosinopril sodium(L-Proline,4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-ox-opropoxy)propox), imidapril,indolapril (Schering, disclosed in J. Cardiovasc. Pharmacol. 5:643, 655(1983)), lisinopril (Merck), losinopril, moexipril, moexiprilhydrochloride (3-Isoquinolinecarboxylic acid,2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,2,3,4-tetrahydro-6,7-dimethoxy-,monohydrochloride, (3S)-CAS RN 82586-52-5), quinapril, quinaprilat,ramipril (Hoechsst) disclosed in EP 79022 and Curr. Ther. Res. 40:74(1986), perindopril erbumine (such as2S,3aS,7aS-1-[(S)—N—[(S)-1-CarboxybutyljalanyljhexahydroA-indolinecarboxylicacid, 1-ethyl ester, compound with tert-butylamine (1:1), e.g., Aceon®,Solvay), perindopril (Servier, disclosed in Eur. J. clin. Pharmacol.31:519 (1987)), quanipril (disclosed in U.S. Pat. No. 4,344,949),spirapril (Schering, disclosed in Acta. Pharmacol. Toxicol. 59 (Supp.5): 173 (1986)), tenocapril, trandolapril, zofenopril (and othersdisclosed in U.S. Pat. No. 4,316,906), rentiapril (fentiapril, disclosedin Clin. Exp. Pharmacol. Physiol. 10:131 (1983)), pivopril, YS980,teprotide (Bradykinin potentiator BPP9a CAS RN 35115-60-7), BRL 36,378(Smith Kline Beecham, see EP80822 and EP60668), MC-838 (Chugai, see CA.102:72588v and Jap. J. Pharmacol. 40:373 (1986), CGS 14824 (Ciba-Geigy,3-([1-ethoxycarbonyl-3-phenyl-(lS)-propyl]amino)-2,3,4,5-tetrahydro-2-ox-o-1-(3 S)-benzazepine-1 aceticacid HCl, see U.K. Patent No. 2103614), CGS 16,617 (Ciba-Geigy,3(S)-[[(1S)-5-amino-1-carboxypentyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-ethanoicacid, see U.S. Pat. No. 4,473,575), Ru 44570 (Hoechst, seeArzneimittelforschung 34:1254 (1985)), R 31-2201 (Hoffman-LaRoche seeFEBS Lett. 165:201 (1984)), CI925 (Pharmacologist 26:243, 266 (1984)),WY-44221 (Wyeth, see J. Med. Chem. 26:394 (1983)), and those disclosedin US2003006922 (paragraph 28), U.S. Pat. Nos. 4,337,201, 4,432,971(phosphonamidates); neutral endopeptidase inhibitors such as omapatrilat(Vanlev®), CGS 30440, cadoxatril and ecadotril, fasidotril (also knownas aladotril or alatriopril), sampatrilat, mixanpril, and gemopatrilat,AVE7688, ER4030, and those disclosed in U.S. Pat. Nos. 5,362,727,5,366,973, 5,225,401, 4,722,810, 5,223,516, 4,749,688, 5,552,397,5,504,080, 5,612,359, 5,525,723, EP0599444, EP0481522, EP0599444,EP0595610, EP0534363, EP534396, EP534492, EP0629627; endothelinantagonists such as tezosentan, A308165, and YM62899, and the like;vasodilators such as hydralazine (apresoline), clonidine (clonidinehydrochloride (1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-,monohydrochloride CAS RN 4205-91-8), catapres, minoxidil (loniten),nicotinyl alcohol (roniacol), diltiazem hydrochloride (such as1,5-Benzothiazepin-4(5H)-one, 3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (+)-cis, e.g., Tiazac®, Forest), isosorbide dinitrate(such as 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate e.g., Isordil®Titradose®, Wyeth-Ayerst), sosorbide mononitrate (such as1,4:3,6-dianhydro-D-glucito-1,5-nitrate, an organic nitrate, e.g.,Ismo®, Wyeth-Ayerst), nitroglycerin (such as 2,3 propanetrioltrinitrate, e.g., Nitrostat® Parke-Davis), verapamil hydrochloride (suchas benzeneacetonitrile, (+)-(alpha)[3-[[2-(3,4 dimethoxypheny1)ethyl]methyl amino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Covera HS® Extended-Release, Searle), chromonar(which may be prepared as disclosed in U.S. Pat. No. 3,282,938),clonitate (Annalen 1870 155), droprenilamine (which may be prepared asdisclosed in DE2521113), lidoflazine (which may be prepared as disclosedin U.S. Pat. No. 3,267,104); prenylamine (which may be prepared asdisclosed in U.S. Pat. No. 3,152,173), propatyl nitrate (which may beprepared as disclosed in French Patent No. 1,103,113), mioflazinehydrochloride (1-Piperazineacetamide,3-(aminocarbonyl)4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6-dichlorophenyl)-,dihydrochloride CAS RN 83898-67-3), mixidine (Benzeneethanamine,3,4-dimethoxy-N-(l-methyl-2-pyrrolidinylidene)-Pyrrolidine,2-[(3,4-dimethoxyphenethyl)imino]-1-methyl-1-Methyl-2-[(3,4-dimethoxyphenethyl)imino]pyrrolidine CAS RN 27737-38-8), molsidomine(1,2,3-Oxadiazolium, 5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, innersalt CAS RN 25717-80-0), isosorbide mononitrate (D-Glucitol,1,4:3,6-dianhydro-, 5-nitrate CAS RN 16051-77-7), erythrityltetranitrate (1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel-CAS RN7297-25-8), clonitrate(1,2-Propanediol, 3-chloro-, dinitrate (7CI, 8CI,9CI) CAS RN 2612-33-1), dipyridamole Ethanol, 2,2′,2″,2′″-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)dinitrilo]tetrakis-CASRN 58-32-2), nicorandil (CAS RN 65141-46-0 3-), pyridinecarboxamide(N-[2-(nitrooxy)ethyl]-Nisoldipine3,5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl esterCAS RN 63675-72-9), nifedipine3,5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN21829-25-4), perhexiline maleate (Piperidine,2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN 6724-53-4),oxprenolol hydrochloride (2-Propanol,1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-, hydrochlorideCAS RN 6452-73-9), pentrinitrol (1,3-Propanediol,2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) CAS RN 1607-17-6),verapamil (Benzeneacetonitrile,α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-CAS RN 52-53-9) and the like; angiotensinII receptor antagonists such as, aprosartan, zolasartan, olmesartan,pratosartan, FI6828K, RNH6270, candesartan(1H-Benzimidazole-7-carboxylic acid,2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]4-yl]methyl]-CAS RN139481-59-7), candesartan cilexetil((+/−)-1-(cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]-1H-benzimidazolecarboxylate, CAS RN 145040-37-5, U.S. Pat. Nos. 5,703,110 and5,196,444), eprosartan(3-[1-4-carboxyphenylmethyl)-2-n-butyl-imidazol-5-yl]-(2-thienylmethyl)propenoic acid, U.S. Pat. Nos. 5,185,351 and 5,650,650), irbesartan(2-n-butyl-3-[[2′-(lh-tetrazol-5-yl)biphenyl-4-yl]methyl]1,3-diazazspiro[4,4]non-1-en-4-one, U.S. Pat. Nos. 5,270,317 and5,352,788), losartan(2-N-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole,potassium salt, U.S. Pat. Nos. 5,138,069, 5,153,197 and 5,128,355),tasosartan(5,8-dihydro-2,4-dimethyl-8-[(2′-(1H-tetrazol-5-yl)[l,r-biphenyl]4-yl)methyl]-pyrido[2,3-d]pyrimidin-7(6H)-one,U.S. Pat. No. 5,149,699), telmisartan(4′-[(1,4-dimethyl-2′-propyl-(2,6′-bi-1H-benzimidazol)-r-yl)]-[1,1′-biphenyl]-2-carboxylicacid, CAS RN 144701-48-4, U.S. Pat. No. 5,591,762), milfasartan,abitesartan, valsartan (Diovan® (Novartis),(S)—N-valeryl-N-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]valine, U.S.Pat. No. 5,399,578), EXP-3137(2-N-butyl-4-chloro-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylicacid, U.S. Pat. Nos. 5,138,069, 5,153,197 and 5,128,355),3-(2′-(tetrazol-5-yl)-l,r-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine,4′[2-ethyl-4-methyl-6-(5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-2-yl]-benzimidazol-1-yl]-methyl]-l,r-biphenyl]-2-carboxylicacid,2-butyl-6-(l-methoxy-1-methylethyl)-2-[2′-)1H-tetrazol-5-yl)biphenyl-4-ylmethyl]guinazolin-4(3H)-one,3-[2′-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H-imidazo[4,5-b]pyridine,2-butyl-4-chloro-1-[(2′-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-carboxylicacid, 2-butyl-4-chloro-1-[[2′-(1H-tetrazol-5-yl) [1,1′-biphenyl]-4-yl]methyl]-1H-imidazole-5-carboxylicacid-1-(ethoxycarbonyl-oxy)ethyl ester potassium salt, dipotassium2-butyl-4-(methylthio)-1-[[2-[[[(propylamino)carbonyl]amino]-sulfonyl](1,1′-biphenyl)-4-yl]methyl]-1H-imidazole-5-carboxylate,methyl-2-[[4-butyl-2-methyl-6-oxo-5-[[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophencarboxylate,5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol-5-ylphenyl)]pyridine,6-butyl-2-(2-phenylethyl)-5[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-methyl]pyrimidin-4-(3H)-one D,Llysine salt,5-methyl-7-n-propyl-8-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-[1,2,4]-triazolo[1,5-c]pyrimidin-2(3H)-one,2,7-diethyl-5-[[2′-(5-tetrazoly)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][1,2,4]triazolepotassium salt,2-[2-butyl-4,5-dihydro-4-oxo-3-[2′-(1H-tetrazol-5-yl)-4-biphenylmethyl]-3H-imidazol[4,5-c]pyridine-5-ylmethyl]benzoicacid, ethyl ester, potassium salt,3-methoxy-2,6-dimethyl-4-[[2′(1H-tetrazol-5-yl)-1,1′-biphenyl-4-yl]methoxy]pyridine,2-ethoxy-1-[[2′-(5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylicacid,1-[N-(2′-(1H-tetrazol-5-yl)biphenyl-4-yl-methyl)-N-valerolylaminomethyl)cyclopentane-1-carboxylicacid, 7-methyl-2n-propyl-3-[[2′1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-6]pyridine,2-[5-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine-3-yl)methyl]-2-quinolinyl]sodiumbenzoate, 2-butyl-6-chloro-4-hydroxymethyl-5-methyl-3-[[2′-(IH-tetrazol-5-yl)biphenyl-4-yl]methyl]pyridine,2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methyl]amino]benzoicacid tetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one,4(S)-[4-(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulfobenzamido)imidazol-1-yl]octanoyl]-L-proline,1-(2,6-dimethylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(H-tetrazol-5-yl)phenyl]-3-pyridinyl]methyl]-2H-imidazol-2-one,5,8-ethano-5,8-dimethyl-2-n-propyl-5,6,7,8-tetrahydro-1-[[2′(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H,4H-1,3,4a,8a-tetrazacyclopentanaphthalene-9-one,4-[1-[2′-(1,2,3,4-tetrazol-5-yl)biphen-4-yl)methylamino]-5,6,7,8-tetrahydro-2-trifylquinazoline,2-(2-chlorobenzoyl)imino-5-ethyl-3-[2′-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl-1,3,4-thiadiazoline,2-[5-ethyl-3-[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-1,3,4-thiazoline-2-ylidene]aminocarbonyl-1-cyclopentencarboxylicacid dipotassium salt, and2-butyl-4-[N-methyl-N-(3-methylcrotonoyl)amino]-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidzole-5-carboxylicacid 1-ethoxycarbonyloxyethyl ester, those disclosed in patentpublications EP475206, EP497150, EP539086, EP539713, EP535463, EP535465,EP542059, EP497121, EP535420, EP407342, EP415886, EP424317, EP435827,EP433983, EP475898, EP490820, EP528762, EP324377, EP323841, EP420237,EP500297, EP426021, EP480204, EP429257, EP430709, EP434249, EP446062,EP505954, EP524217, EP514197, EP514198, EP514193, EP514192, EP450566,EP468372, EP485929, EP503162, EP533058, EP467207 EP399731, EP399732,EP412848, EP453210, EP456442, EP470794, EP470795, EP495626, EP495627,EP499414, EP499416, EP499415, EP511791, EP516392, EP520723, EP520724,EP539066, EP438869, EP505893, EP530702, EP400835, EP400974, EP401030,EP407102, EP411766, EP409332, EP412594, EP419048, EP480659, EP481614,EP490587, EP467715, EP479479, EP502725, EP503838, EP505098, EP505111EP513,979 EP507594, EP510812, EP511767, EP512675, EP512676, EP512870,EP517357, EP537937, EP534706, EP527534, EP540356, EP461040, EP540039,EP465368, EP498723, EP498722, EP498721, EP515265, EP503785, EP501892,EP519831, EP532410, EP498361, EP432737, EP504888, EP508393, EP508445,EP403159, EP403158, EP425211, EP427463, EP437103, EP481448, EP488532,EP501269, EP500409, EP540400, EP005528, EP028834, EP028833, EP411507,EP425921, EP430300, EP434038, EP442473, EP443568, EP445811, EP459136,EP483683, EP518033, EP520423, EP531876, EP531874, EP392317, EP468470,EP470543, EP502314, EP529253, EP543263, EP540209, EP449699, EP465323,EP521768, EP415594, WO92/14468, WO93/08171, WO93/08169, WO91/00277,WO91/00281, WO91/14367, WO92/00067, WO92/00977, WO92/20342, WO93/04045,WO93/04046, WO91/15206, WO92/14714, WO92/09600, WO92/16552, WO93/05025,WO93/03018, WO91/07404, WO92/02508, WO92/13853, WO91/19697, WO91/11909,WO91/12001, WO91/11999, WO91/15209, WO91/15479, WO92/20687, WO92/20662,WO92/20661, WO93/01177, WO91/14679, WO91/13063, WO92/13564, WO91/17148,WO91/18888, WO91/19715, WO92/02257, WO92/04335, WO92/05161, WO92/07852,WO92/15577, WO93/03033, WO91/16313, WO92/00068, WO92/02510, WO92/09278,WO9210179, WO92/10180, WO92/10186, WO92/10181, WO92/10097, WO92/10183,WO92/10182, WO92/10187, WO92/10184, WO92/10188, WO92/10180, WO92/10185,WO92/20651, WO93/03722, WO93/06828, WO93/03040, WO92/19211, WO92/22533,WO92/06081, WO92/05784, WO93/00341, WO92/04343, WO92/04059, U.S. Pat.Nos. 5,104,877, 5,187,168, 5,149,699, 5,185,340, 4,880,804, 5,138,069,4,916,129, 5,153,197, 5,173,494, 5,137,906, 5,155,126, 5,140,037,5,137,902, 5,157,026, 5,053,329, 5,132,216, 5,057,522, 5,066,586,5,089,626, 5,049,565, 5,087,702, 5,124,335, 5,102,880, 5,128,327,5,151,435, 5,202,322, 5,187,159, 5,198,438, 5,182,288, 5,036,048,5,140,036, 5,087,634, 5,196,537, 5,153,347, 5,191,086, 5,190,942,5,177,097, 5,212,177, 5,208,234, 5,208,235, 5,212,195, 5,130,439,5,045,540, 5,041,152, and 5,210,204, and pharmaceutically acceptablesalts and esters thereof, a/f3 adrenergic blockers such as nipradilol,arotinolol, amosulalol, bretylium tosylate (CAS RN: 61-75-6),dihydroergtamine mesylate (such as ergotaman-3′,6′, 18-trione,9,10-dihydro-12′-hydroxy-2′-methyl-5′-(phenylmethyl)-, (5′(α))-,monomethanesulfonate, e.g., DHE 45® Injection, Novartis), carvedilol(such as (+)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol, e.g., Coreg®, SmithKline Beecham), labetalol (such as5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino]ethyljsalicylamidemonohydrochloride, e.g., Normodyne®, Schering), bretylium tosylate(Benzenemethanaminium, 2-bromo-N-ethyl-N,N-dimethyl-, salt with4-methylbenzenesulfonic acid (1:1) CAS RN 61-75-6), phentolaminemesylate (Phenol,3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-,monomethanesulfonate (salt) CAS RN 65-28-1), solypertine tartrate(5H-1,3-Dioxolo[4,5-f]indole,7-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-,(2R,3R)-2,3-dihydroxybutanedioate (1:1) CAS RN 5591-43-5), zolertinehydrochloride (Piperazine, 1-phenyl4-[2-(1H-tetrazol-5-yl)ethyl]-,monohydrochloride (8Cl, 9Cl) CAS RN 7241-94-3) and the like; aadrenergic receptor blockers, such as alfuzosin (CAS RN: 81403-68-1),terazosin, urapidil, prazosin (Minipress®), tamsulosin, bunazosin,trimazosin, doxazosin, naftopidil, indoramin, WHP 164, XENOlO,fenspiride hydrochloride (which may be prepared as disclosed in U.S.Pat. No. 3,399,192), proroxan (CAS RN 33743-96-3), and labetalolhydrochloride and combinations thereof; a 2 agonists such as methyldopa,methyldopa HCL, lofexidine, tiamenidine, moxonidine, rilmenidine,guanobenz, and the like; aldosterone inhibitors, and the like; renininhibitors including Aliskiren (SPPlOO; Novartis/Speedel);angiopoietin-2-binding agents such as those disclosed in WO03/030833;anti-angina agents such as ranolazine (hydrochloride1-Piperazineacetamide,N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6), betaxolol hydrochloride (2-Propanol,1-[4-[2 (cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-,hydrochloride CAS RN 63659-19-8), butoprozine hydrochloride (Methanone,[4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-,monohydrochloride CAS RN 62134-34-3), cinepazetmaleatel-Piperazineacetic acid,4-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-, ethyl ester,(2Z)-2-butenedioate (1:1) CAS RN 50679-07-7), tosifen(Benzenesulfonamide, 4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl] amino]carbonyl]-CAS RN 32295-184), verapamilhydrochloride(Benzeneacetonitrile,α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-,monohydrochloride CAS RN 152-114), molsidomine (1,2,3-Oxadiazolium,5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN25717-80-0), and ranolazine hydrochloride (1-Piperazineacetamide,N-(2,6-dimethylphenyl)4-[2-hydroxy-3-(2-meth-oxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6); tosifen (Benzenesulfonamide,4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN32295-184); adrenergic stimulants such as guanfacine hydrochloride (suchas N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride, e.g.,Tenex® Tablets available from Robins); methyldopa-hydrochlorothiazide(such as levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined withHydrochlorothiazide (such as6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide1,1-dioxide, e.g., the combination as, e.g., Aldoril® Tablets availablefrom Merck), methyldopa-chlorothiazide (such as 6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide and methyldopa asdescribed above, e.g., Aldoclor®, Merck), clonidine hydrochloride (suchas 2-(2,6-dichlorophenyl amino)-2-imidazoline hydrochloride andchlorthalidone (such as 2-chloro-5-(1-hydroxy-3-oxo-1-isoindolinyl)benzenesulfonamide), e.g., Combipres®, Boehringer Ingelheim), clonidinehydrochloride (such as 2-(2,6-dichlorophenylamino)-2-imidazolinehydrochloride, e.g., Catapres®, Boehringer Ingelheim), clonidine(1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-CAS RN4205-90-7), Hyzaar (Merck; a combination of losartan andhydrochlorothiazide), Co-Diovan (Novartis; a combination of valsartanand hydrochlorothiazide, Lotrel (Novartis; a combination of benazepriland amlodipine) and Caduet (Pfizer; a combination of amlodipine andatorvastatin), and those agents disclosed in US20030069221.

1.2.2.11 Agents for the Treatment of Respiratory Disorders

The GCC agonist peptides described herein can be used in combinationtherapy with one or more of the following agents useful in the treatmentof respiratory and other disorders including but not limited to: (1)β-agonists including but not limited to: albuterol (PRO VENTIL®, S ALBUTAMOl®, VENTOLIN®), bambuterol, bitoterol, clenbuterol, fenoterol,formoterol, isoetharine (BRONKOSOL®, BRONKOMETER®), metaproterenol(ALUPENT®, METAPREL®), pirbuterol (MAXAIR®), reproterol, rimiterol,salmeterol, terbutaline (BRETHAIRE®, BRETHINE®, BRICANYL®), adrenalin,isoproterenol (ISUPREL®), epinephrine bitartrate (PRIMATENE®),ephedrine, orciprenline, fenoterol and isoetharine; (2) steroids,including but not limited to beclomethasone, beclomethasonedipropionate, betamethasone, budesonide, bunedoside, butixocort,dexamethasone, flunisolide, fluocortin, fluticasone, hydrocortisone,methyl prednisone, mometasone, predonisolone, predonisone, tipredane,tixocortal, triamcinolone, and triamcinolone acetonide; (3)02-agonist-corticosteroid combinations [e.g., salmeterol-fluticasone (ADV AIR®), formoterol-budesonid (S YMBICORT®)]; (4) leukotriene D4receptor antagonists/leukotriene antagonists/LTD4 antagonists (i.e., anycompound that is capable of blocking, inhibiting, reducing or otherwiseinterrupting the interaction between leukotrienes and the Cys LTIreceptor) including but not limited to: zafhiukast, montelukast,montelukast sodium (SINGULAIR®), pranlukast, iralukast, pobilukast,SKB-106,203 and compounds described as having LTD4 antagonizing activitydescribed in U.S. Pat. No. 5,565,473; (5) 5-lipoxygenase inhibitorsand/or leukotriene biosynthesis inhibitors [e.g., zileuton and BAY1005(CA registry 128253-31-6)]; (6) histamine H1 receptorantagonists/antihistamines (i.e., any compound that is capable ofblocking, inhibiting, reducing or otherwise interrupting the interactionbetween histamine and its receptor) including but not limited to:astemizole, acrivastine, antazoline, azatadine, azelastine, astamizole,bromopheniramine, bromopheniramine maleate, carbinoxamine, carebastine,cetirizine, chlorpheniramine, chloropheniramine maleate, cimetidineclemastine, cyclizine, cyproheptadine, descarboethoxyloratadine,dexchlorpheniramine, dimethindene, diphenhydramine, diphenylpyraline,doxylamine succinate, doxylarnine, ebastine, efletirizine, epinastine,famotidine, fexofenadine, hydroxyzine, hydroxyzine, ketotifen,levocabastine, levocetirizine, levocetirizine, loratadine, meclizine,mepyramine, mequitazine, methdilazine, mianserin, mizolastine,noberastine, norasternizole, noraztemizole, phenindamine, pheniramine,picumast, promethazine, pynlamine, pyrilamine, ranitidine, temelastine,terfenadine, trimeprazine, tripelenamine, and triprolidine; (7) ananticholinergic including but not limited to: atropine, benztropine,biperiden, flutropium, hyoscyamine (e.g. Levsin®; Levbid®; Levsin/SL®,Anaspaz®, Levsinex Timecaps®, NuLev®), ilutropium, ipratropium,ipratropium bromide, methscopolamine, oxybutinin, rispenzepine,scopolamine, and tiotropium; (8) an anti-tussive including but notlimited to: dextromethorphan, codeine, and hydromorphone; (9) adecongestant including but not limited to: pseudoephedrine andphenylpropanolamine; (10) an expectorant including but not limited to:guafenesin, guaicolsulfate, terpin, ammonium chloride, glycerolguaicolate, and iodinated glycerol; (11) a bronchodilator including butnot limited to: theophylline and aminophylline; (12) ananti-inflammatory including but not limited to: fluribiprofen,diclophenac, indomethacin, ketoprofen, S-ketroprophen, tenoxicam; (13) aPDE (phosphodiesterase) inhibitor including but not limited to thosedisclosed herein; (14) a recombinant humanized monoclonal antibody [e.g.xolair (also called omalizumab), rhuMab, and talizumab]; (15) ahumanized lung surfactant including recombinant forms of surfactantproteins SP-B, SP-C or SP-D [e.g. SURFAXIN®, formerly known as dsc-104(Discovery Laboratories)], (16) agents that inhibit epithelial sodiumchannels (ENaC) such as amiloride and related compounds; (17)antimicrobial agents used to treat pulmonary infections such asacyclovir, amikacin, amoxicillin, doxycycline, trimethoprinsulfamethoxazole, amphotericin B, azithromycin, clarithromycin,roxithromycin, clarithromycin, cephalosporins (ceffoxitin, cefmetazoleetc), ciprofloxacin, ethambutol, gentimycin, ganciclovir, imipenem,isoniazid, itraconazole, penicillin, ribavirin, rifampin, rifabutin,amantadine, rimantidine, streptomycin, tobramycin, and vancomycin; (18)agents that activate chloride secretion through Ca++ dependent chloridechannels (such as purinergic receptor (P2Y(2) agonists); (19) agentsthat decrease sputum viscosity, such as human recombinant DNase 1,(Pulmozyme®); (20) nonsteroidal anti-inflammatory agents (acemetacin,acetaminophen, acetyl salicylic acid, alclofenac, alminoprofen, apazone,aspirin, benoxaprofen, bezpiperylon, bucloxic acid, carprofen, clidanac,diclofenac, diclofenac, diflunisal, diflusinal, etodolac, fenbufen,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, flufenisal, fluprofen, flurbiprofen,flurbiprofen, furofenac, ibufenac, ibuprofen, indomethacin,indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen,ketorolac, meclofenamic acid, meclofenamic acid, mefenamic acid,mefenamic acid, miroprofen, mofebutazone, nabumetone oxaprozin,naproxen, naproxen, niflumic acid, oxaprozin, oxpinac, oxyphenbutazone,phenacetin, phenylbutazone, phenylbutazone, piroxicam, piroxicam,pirprofen, pranoprofen, sudoxicam, tenoxican, sulfasalazine, sulindac,sulindac, suprofen, tiaprofenic acid, tiopinac, tioxaprofen, tolfenamicacid, tolmetin, tolmetin, zidometacin, zomepirac, and zomepirac); and(21) aerosolized antioxidant therapeutics such as S-Nitrosoglutathione.

1.2.2.12 Anti-Diabetic Agents

The GCC agonist peptides described herein can be used in therapeuticcombination with one or more anti-diabetic agents, including but notlimited to: PPARγ agonists such as glitazones (e.g., WAY-120,744, AD5075, balaglitazone, ciglitazone, darglitazone (CP-86325, Pfizer),englitazone (CP-68722, Pfizer), isaglitazone (MIT/J&J), MCC-555(Mitsibishi disclosed in U.S. Pat. No. 5,594,016), pioglitazone (such assuch as Actos™ pioglitazone; Takeda), rosiglitazone (Avandia™; SmithKline Beecham), rosiglitazone maleate, troglitazone (Rezulin®, disclosedin U.S. Pat. No. 4,572,912), rivoglitazone (CS-Ol 1, Sankyo), GL-262570(Glaxo Welcome), BRL49653 (disclosed in WO98/05331), CLX-0921, 5-BTZD,GW-0207, LG-100641, JJT-501 (JPNT/P&U), L-895645 (Merck), R-119702(Sankyo/Pfizer), NN-2344 (Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512,LY-519818, R483 (Roche), T131 (Tularik), and the like and compoundsdisclosed in U.S. Pat. Nos. 4,687,777, 5,002,953, 5,741,803, 5,965,584,6,150,383, 6,150,384, 6,166,042, 6,166,043, 6,172,090, 6,211,205,6,271,243, 6,288,095, 6,303,640, 6,329,404, 5,994,554, WO97/10813,WO97/27857, WO97/28115, WO97/28137, WO97/27847, WO00/76488, WO03/000685,WO03/027112, WO03/035602, WO03/048130, WO03/055867, and pharmaceuticallyacceptable salts thereof; biguanides such as metformin hydrochloride(N,N-dimethylimidodicarbonimidic diamide hydrochloride, such asGlucophage™, Bristol-Myers Squibb); metformin hydrochloride withglyburide, such as Glucovance™, Bristol-Myers Squibb); buformin(Imidodicarbonimidic diamide, N-butyl-); etoformine(1-Butyl-2-ethylbiguanide, Schering A. G.); other metformin salt forms(including where the salt is chosen from the group of, acetate,benzoate, citrate, ftimarate, embonate, chlorophenoxyacetate, glycolate,palmoate, aspartate, methanesulphonate, maleate,parachlorophenoxyisobutyrate, formate, lactate, succinate, sulphate,tartrate, cyclohexanecarboxylate, hexanoate, octanoate, decanoate,hexadecanoate, octodecanoate, benzenesulphonate, trimethoxybenzoate,paratoluenesulphonate, adamantanecarboxylate, glycoxylate, glutarnate,pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucosephosphate,nitrate, sulphite, dithionate and phosphate), and phenformin; proteintyrosine phosphatase-IB (PTP-IB) inhibitors, such as A-401,674, KR61639, OC-060062, OC-83839, OC-297962, MC52445, MC52453, ISIS 113715,and those disclosed in WO99/585521, WO99/58518, WO99/58522, WO99/61435,WO03/032916, WO003/032982, WO003/041729, WO003/055883, WO02/26707,WO02/26743, JP2002114768, and pharmaceutically acceptable salts andesters thereof; sulfonylureas such as acetohexamide (e.g. Dymelor, EliLilly), carbutamide, chlorpropamide (e.g. Diabinese®, Pfizer),gliamilide (Pfizer), gliclazide (e.g. Diamcron, Servier Canada Inc),glimepiride (e.g. disclosed in U.S. Pat. No. 4,379,785, such as Amaryl,Aventis), glipentide, glipizide (e.g. Glucotrol or Glucotrol XL ExtendedRelease, Pfizer), gliquidone, glisolamide, glyburide/glibenclamide (e.g.Micronase or Glynase Prestab, Pharmacia & Upjohn and Diabeta, Aventis),tolazamide (e.g. Tolinase), and tolbutamide (e.g. Orinase), andpharmaceutically acceptable salts and esters thereof; meglitinides suchas repaglinide (e.g. Pranidin®, Novo Nordisk), KAD1229 (PF/Kissei), andnateglinide (e.g. Starlix®, Novartis), and pharmaceutically acceptablesalts and esters thereof; a glucoside hydrolase inhibitors (or glucosideinhibitors) such as acarbose (e.g. Precose™, Bayer disclosed in U.S.Pat. No. 4,904,769), miglitol (such as GLYSET™, Pharmacia & Upjohndisclosed in U.S. Pat. No. 4,639,436), camiglibose (Methyl6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-alpha-D-glucopyranoside,Marion Merrell Dow), voglibose (Takeda), adiposine, emiglitate,pradimicin-Q, salbostatin, CKD-711, MDL-25,637, MDL-73,945, and MOR 14,and the compounds disclosed in U.S. Pat. Nos. 4,062,950, 4,174,439,4,254,256, 4,701,559, 4,639,436, 5,192,772, 4,634,765, 5,157,116,5,504,078, 5,091,418, 5,217,877, US51091 and WOO 1/47528 (polyamines);α-amylase inhibitors such as tendamistat, trestatin, and Al-3688, andthe compounds disclosed in U.S. Pat. Nos. 4,451,455, 4,623,714, and4,273,765; SGLT2 inhibtors including those disclosed in U.S. Pat. Nos.6,414,126 and 6,515,117; an aP2 inhibitor such as disclosed in U.S. Pat.No. 6,548,529; insulin secreatagogues such as linogliride, A-4166,forskilin, dibutyrl cAMP, isobutylmethylxanthine (IBMX), andpharmaceutically acceptable salts and esters thereof; fatty acidoxidation inhibitors, such as clomoxir, and etomoxir, andpharmaceutically acceptable salts and esters thereof; A2 antagonists,such as midaglizole, isaglidole, deriglidole, idazoxan, earoxan, andfluparoxan, and pharmaceutically acceptable salts and esters thereof;insulin and related compounds (e.g. insulin mimetics) such as biota,LP-100, novarapid, insulin detemir, insulin lispro, insulin glargine,insulin zinc suspension (lente and ultralente), Lys-Pro insulin, GLP-I(1-36) amide, GLP-I (73-7) (insulintropin, disclosed in U.S. Pat. No.5,614,492), LY-315902 (Lilly), GLP-I (7-36)-NH2), AL-401 (Autoimmune),certain compositions as disclosed in U.S. Pat. Nos. 4,579,730,4,849,405, 4,963,526, 5,642,868, 5,763,396, 5,824,638, 5,843,866,6,153,632, 6,191,105, and WO 85/05029, and primate, rodent, or rabbitinsulin including biologically active variants thereof including allelicvariants, more preferably human insulin available in recombinant form(sources of human insulin include pharmaceutically acceptable andsterile formulations such as those available from Eli Lilly(Indianapolis, Ind. 46285) as Humulin™ (human insulin rDNA origin), alsosee the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) MedicalEconomics, Thomson Healthcare (disclosing other suitable humaninsulins); non-thiazolidinediones such as JT-501 and farglitazar(GW-2570/GI-262579), and pharmaceutically acceptable salts and estersthereof; PPARα/γ dual agonists such as AR-HO39242 (Aztrazeneca),GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940, GW-1536, GW-1929,GW-2433, KRP-297 (Kyorin Merck; 5-[(2,4-Dioxo thiazolidinyl)methyl]methoxy-N-[[4-(trifluoromethyl)phenyl] methyljbenzamide), L-796449,LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar (BMS),tesaglitzar (Astrazeneca), reglitazar (JTT-501) and those disclosed inWO99/16758, WO99/19313, WO99/20614, WO99/38850, WO00/23415, WO00/23417,WO00/23445, WO00/50414, WOO1/00579, WOO1/79150, WOO2/062799,WOO3/004458, WOO3/016265, WOO3/018010, WOO3/033481, WOO3/033450,WOO3/033453, WOO3/043985, WO 031053976, U.S. application Ser. No.09/664,598, filed Sep. 18, 2000, Murakami et al. Diabetes 47, 1841-1847(1998), and pharmaceutically acceptable salts and esters thereof; otherinsulin sensitizing drugs; VPAC2 receptor agonists; GLK modulators, suchas those disclosed in WO03/015774; retinoid modulators such as thosedisclosed in WO03/000249; GSK 30/GSK 3 inhibitors such as4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine andthose compounds disclosed in WO03/024447, WO03/037869, WO03/037877,WOO3/037891, WOO3/068773, EP1295884, EP1295885, and the like; glycogenphosphorylase (HGLPa) inhibitors such as CP-368,296, CP-316,819,BAYR3401, and compounds disclosed in WOO 1/94300, WOO2/20530,WOO3/037864, and pharmaceutically acceptable salts or esters thereof,ATP consumption promotors such as those disclosed in WO03/007990; TRB3inhibitors; vanilloid receptor ligands such as those disclosed inWOO3/049702; hypoglycemic agents such as those disclosed in WOO3/015781and WO03/040114; glycogen synthase kinase 3 inhibitors such as thosedisclosed in WOO3/035663 agents such as those disclosed in WO99/51225,US20030134890, WO01/24786, and WO03/059870; insulin-responsive DNAbinding protein-1 (IRDBP-I) as disclosed in WO03/057827, and the like;adenosine A2 antagonists such as those disclosed in WOO3/035639,WO03/035640, and the like; PPARδ agonists such as GW 501516, GW 590735,and compounds disclosed in JP10237049 and WOO2/14291; dipeptidylpeptidase IV (DP-IV) inhibitors, such as isoleucine thiazolidide,NVP-DPP728A (1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl] amino]acetyl]-2-cyano-(S)-pyrrolidine, disclosed by Hughes et al,Biochemistry, 38(36), 11597-11603, 1999), P32/98, NVP-LAF-237, P3298,TSL225 (tryptophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid,disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)1537-1540), valine pyrrolidide, TMC-2A/2B/2C, CD-26 inhibitors,FE999011, P9310/K364, VIP 0177, DPP4, SDZ 274-444, 2-cyanopyrrolididesand 4-cyanopyrrolidides as disclosed by Ashworth et al, Bioorg. & Med.Chem. Lett., Vol. 6, No. 22, pp 1163-1166 and 2745-2748 (1996), and thecompounds disclosed in U.S. Pat. Nos. 6,395,767, 6,573,287, 6,395,767(compounds disclosed include BMS-477118, BMS-471211 and BMS 538,305),WO99/38501, WO99/46272, WO99/67279, WO99/67278, WO99/61431 WO03/004498,WO03/004496, EP1258476, WO02/083128, WO02/062764, WO03/000250,WO03/002530, WO03/002531, WO03/002553, WO03/002593, WO03/000180, andWO03/000181; GLP-I agonists such as exendin-3 and exendin-4 (includingthe 39 aa polypeptide synthetic exendin-4 called Exenatide®), andcompounds disclosed in US2003087821 and NZ 504256, and pharmaceuticallyacceptable salts and esters thereof; peptides including amlintide andSymlin® (pramlintide acetate); and glycokinase activators such as thosedisclosed in US2002103199 (fused heteroaromatic compounds) andWO02/48106 (isoindolin-1-one-substituted propionamide compounds).

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties. However, where apatent, patent application, or publication containing expressdefinitions is incorporated by reference, those express definitionsshould be understood to apply to the incorporated patent, patentapplication, or publication in which they are found, and not to theremainder of the text of this application. In the case of conflict, thepresent specification, including definitions, will control. Thereferences cited herein are not admitted to be prior art to theinvention.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention. It will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the invention, and further that otheraspects, advantages and modifications will be apparent to those skilledin the art to which the invention pertains.

EXAMPLES Example 1: Preparation of Side-Chain Protected Fragments ofSP-304

Attachment Fmoc-AA-OH to 2-C1Trt Resin

[162] 2-ClTrt resin (10 g, substitution=1.0 mmol/g resin) was suspendedin 100 mL of dichloromethane (DCM) for 5 minutes, and then drained. Theesterification was performed using 1.5 equiv. of Fmoc-amino acid and 1.7equiv. Diisopropylethylamine (DIEA) in 80 mL of DCM (with minimumquantity of dimethylformamide (DMF) to dissolve the amino acidcompletely) for 2 hours. The resulting resin was washed with 60 mL ofDCM and endcapped with 60 mL of DIEA/methanol (1:9, v/v) solution for 30minutes. The loaded resin was then washed with DCM (6 vol.) for 2 times,DMF (6 vol.) for 3 times and methyl t-butylether (MTBE) (6 vol.) for 3times, and dried under high vacuum. The substitution of theFmoc-protected resin was determined by Fmoc release assay. Finally, theFmoc group was deprotected with a mixture of 5% piperidine, 1%1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) and 1% N-Hydroxybenzotriazole(HOBt) in DMF (10 vol.) for 2 times and the resin was washed and driedunder high vacuum to give the final resin for peptide synthesis. Theresults of the experiments are listed in Table VIII below.

TABLE VIII Preparation of H-Gly-2ClTrt and H-Leu-2ClTrt resinSubstitution of Synthesis the loaded Amino acid 2-ClTrt scale resin fromFmoc Yield resin (mmol) release assay (Weight, % yield) H-Gly-2ClTrtresin 4.16 0.57 mmol/g resin    6 g, 82% H-Leu-2ClTrt resin 5.20 0.81mmol/g resin  6.4 g, 100% H-Gly-2ClTrt resin 300 0.80 mmol/g resin 328.6g, 88% H-Leu-2ClTrt resin 300 0.75 mmol/g resin 338.5 g, 84%

Synthesis of Side-Chain-Protected Fragments A and B

H-Gly-2-C1Trt resin or H-Leu-2-C1Trt resin was suspended in DMF (10vol.) for 20 minutes, then drained. The resulting resin was washed withDMF (10 vol.) for 5 minutes. The chain assembly was conducted using thestandard Fmoc chemistry. Generally, 1.5 equiv. of Fmoc amino acid and1.5 equiv. of HOBt were dissolved in DMF (4.5 vol.), followed byaddition of 1.5 equiv. of DIEA. Then, the resulting solution was cooledto below 5° C. with an ice water bath, and activated by addition of 1.5equiv. of HBTU. DCM (1.5 vol.) was added to the resin, followed byaddition of the activated Fmoc amino acid solution. The resultingmixture was stirred at room temperature for 2 hours and the completionof the acylation was monitored by Kaiser Test. If Kaiser Test indicatedthe presence of unreacted amine after 2 hours, recoupling with the sameprotocol using 1.0 equiv. of Fmoc amino acid, 1.0 equiv. of HOBt and 1.0equiv. of DIEA was required. Capping was generally achieved byacetylating the unreacted amine with a mixture of aceticanhydride/pyridine/DMF solution. The peptide sequence was assembled byrepeating the above capping procedure with the corresponding Fmoc-aminoacid derivatives in the sequence from C- to N-terminal. The coupling ofFmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH residue was achieved by using 2.0equiv. of Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH, 2.0 of equiv. HOBt and2.0 equiv. of DIC in situ activation in DCM/DMF protocol to minimizeracemization of cysteine.

After completion of the synthetic step, the peptide resin was thoroughlywashed with DMF (10 vol.), MTBE (10 vol.), DMF (10 vol. 3 times) andMTBE (10 vol. 3 times) and subsequently dried in a vacuum oven to aconstant weight.

The side-chain-protected peptide was cleaved from the resin using 1%TFA/DCM (10 vol.) for 3 times, 5 minutes for each time and the cleavagefractions were collected onto pyridine each time (1:1 volume ratio toTFA in each cleavage fraction). The peptide resin was washed with DCM(7.5 vol.). The fractions were combined and concentrated under vacuum to10% of the original volume, and the resulting solution was reconstitutedwith ethanol (3 vol.) and concentrated to 50% of the original volume.Finally, the peptide was precipitated out by addition of water (1 vol.).The solid was collected by vacuum filtration or centrifugation andwashed with water twice. The product was dried in vacuum to a constantweight and subjected to HPLC and ES-MS analysis. The results of theexperiments are presented in Table IX below.

TABLE IX Preparation of Fragments A and B Final peptide resin weight andYield of Synthesis yield from the Fragment scale weight gain of obtainedPurity Fragment Masses Fragment (mmol) the resin (% yield) (HPLC)Calculated/Found* FmocAA7-14OH (1) 3.4  9.5 g, 66.4% 3.337 g 87.8%1599.95/1598.86 (61.3%) BocAA1-6OH (2) 5.2  11.8 g, 76.3% 5.896 g 94.6%1474.80/1473.94 (76.9%) FmocAA7-14OH (1′) 200 479.7 g, 74.4% 213.7 g90.1% 1599.95/1598.81 (66.8%) BocAA1-6OH (2′) 200 544.0 g, 101.3% 247.0g 94.2% 1474.80/1473.94 (83.7%) *Calculated = average molecular mass;Found = mono-isotopic mass by ES-MS

Example 2: Condensation of Fragments of Sp-304 in Solution

Synthesis of Fragment C: H-AA15-16OtBu (1-1)

A solution of Fmoc-Cys(Acm)-OH (124.38 gm, 0.3 mol), H-Leu-OtBu.HCl(67.12 gm, 0.3 mol), and HOBt (40.54 gm, 0.3 mol) in DMF (600 mL) wascooled to −5° C. 2-[1H-Benzotriazole-1-yl]-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) (113.79 gm, 0.3 mol) was added and dissolvedcompletely. DIEA (183.1 mL, 1.05 mol) was added dropwise over a periodof 105 minutes at the same temperature with good stirring, keeping thepH of the mixture between 6 and 7. Stirring was continued for 15 minutesat 0° C. and the reaction was monitored with TLC. The reaction mixturewas diluted with ethyl acetate (EtOAc) (600 mL) and 5% H₃PO₄ (300 mL).The organic layer was separated and the aqueous layer was extracted withEtOAc (600 mL). The combined extracts were washed with 5% H₃PO₄ (2times), H₂O (1 time), saturated NaHCO₃ (3 times), H₂O (2 times), andbrine (2 times). The solution was dried over MgSO₄ anhydrous, filtered,and evaporated in vacuo to dryness. The product was recrystallized frompetroleum ether/EtOAc (3:1) and dried: 166.75 gm (yield 95.0%, purity99.0%).

Fmoc-Cys(Acm)-Leu-OtBu (166.75 gm, 0.277 mol) was dissolved in a 10%piperidine/DCM solution (810 mL) with stirring. The reaction wasmonitored with TLC. After the reaction was completed in 3 hours, thesolvent and the volatile materials were removed using a rotavap. Theoily material obtained was triturated with petroleum ether to remove theby-products by decantation. The residue, a syrup, was taken up in EtOAc,and washed with a mixture solution of NaH₂PO₄/Na₂HPO₄ (pH=6), thensaturated NaHCO₃, purified H₂O, and brine. The organic layer was driedover MgSO₄ anhydrous. Evaporation of the solvent and the volatilematerials yielded an oily product H-Cys(Acm)-Leu-OtBu (1-1) (73.23 gm,yield: 73.1%, purity: 98.0%).

Synthesis of Fragment B-C: HAA7-16OtBu (2-3)

A solution of Fmoc-AA7-140H (1′) (198.3 gm, 124.0 mmol), H-AA15-16OtBu(1-1) (52.3 gm, 148.8 mmol) and Cl-HOBt (21.0 gm, 124.0 mmol) in DMF(2500 mL) was cooled to −5° C. HBTU (51.7 gm, 136.4 mmol) was added anddissolved completely. DIEA (54.1 mL, 310 mmol) was then added withstirring, keeping the pH of the mixture between 6 and 7. Stirring wascontinued for 30 minutes at 0-5° C. The reaction mixture was allowed towarm up to 20-27° C. slowly and stirring was continued for one and ahalf hours. Then, the mixture was poured into precooled (10-20° C.) 0.5N HCl aq. (20 L). The suspension was stored at 20-25° C. for 45 minutes.The solid was collected by filtering the suspension through afritted-glass funnel of medium porosity and subsequent washing with 0.5N HCl aq (3500 mL, 2 times), purified water (3500 mL), saturated NaHCO₃aq. (3500 mL, 2 times) and purified water (3500 mL, 2 times) and diethylether (2000 mL, 2 times). Finally, the wet, crude peptide FmocAA7-16OtBu(2-3) was dried in a desiccator under high vacuum at room temperature toyield product 238.22 gm (purity, 85.27%, yield 98.9%).

Synthesis of the Fully Protected A-B-C: BocAA1-16OtBu (3-3)

A solution of HAA7-16OtBu (2-3) (183.67 gm, 106.71 mmol), BocAA1-6-OH(2′) (157.65 gm, 106.71 mmol), and 6-Cl-HOBt (18.141 gm, 106.71 mmol) inDMF (3 L) were cooled to at −3 to 0° C. HBTU (44.523 gm, 117.38 mmol)was added and dissolved completely. DIEA (55.8 mL, 320.13 mmol) was thenadded with stirring, keeping the pH of the mixture 6. Stirring wascontinued for 20 minutes at −5 to 0° C. The reaction mixture was allowedto warm up to 25° C. slowly and stirring was continued for 2.5 hours,followed by further addition of HBTU (4.048 gm, 10.671 mmol) and DIEA (2mL). Stirring was continued for another 1.5 hours. The resulting mixturewas poured into MeOH (15 L), and the precipitate was collected andwashed with MeOH/DMF mixture (5:1, v/v) (2 times, 3 L), 0.1N HCl (3 L, 2times), saturated NaHCO₃ (2 times), purified water (3 times), diethylether (2 times) and dried in vacuo to yield the product BocAA1-16OtBu(3-3) (278.0 gm, yield 82.0%. Note: the purity was determined after thedeprotection).

Synthesis of the Partially Protected Linear SP-304: HAA1-160H (4-4)

A mixture of TFA/TIS/EDT (8:1:1, 2400 mL) was cooled to (0-5° C.) undernitrogen and Boc-AA1-16OtBu (3-3) (201 gm) was added in portions. Theresulting suspension was stirred at 0-10° C. for 30 min, then thereaction solution was allowed to warm up to 20-25° C. with a water bath(10 minutes) and stirring was continued for additional 1 hr and 50 minat the same temperature. The reaction mixture was poured into pre-cooled(10° C.) MTBE (18 L). Some heat was evolved during the addition of thepeptide/TFA solution and the internal temperature went up to 25° C. Theresulting suspension was then stored in an ice-water bath (5-10° C.) for40 min. The precipitate was collected by filtration and washed with MTBE(2000 mL, 4 times), and dried in vacuum over P₂O₅, yielding 148.37 gm ofoff-white product, HAA1-160H (4-4) (purity: 62.23%, ES-MS, MW:calculated=1828.07, found=1826.67).

Example 3: Oxidative Cyclization and Purification of SP-304 byPolystyrenic Absorbent Resin

HAA1-160H (4-4) (0.58 gm) was dissolved in 5 mL of acetonitrile anddiluted with 575 mL of purified water. The solution was adjusted to pH8-9 with 25% ammonia solution, and 3% hydrogen peroxide (0.58 mL) wasadded, then the reaction mixture was kept for an hour with monitoringthe disulfide formation by HPLC. Nitrogen was then passed through thereaction mixture and the solution was acidified to pH 3-4 with aceticacid (71.8% HPLC, recovery 98.5% estimated from peak area). Theresulting mixture was added 1% iodine/ACN dropwise over a period of 10minutes with good stirring until the yellow color of the iodinepersisted. Stirring was continued for 30 minutes at 17-20° C. The iodinewas quenched by addition of 0.5 M ascorbic acid aq. until the yellowdisappeared. Then, the pH of the mixture was adjusted to 6-7 with 25%ammonia solution (51.0% HPLC, recovery 50% estimated from peak area).

The polystyrenic absorbent resin (D101) was packed to a 3(ID)×9(L) CMcolumn and well equilibrated with 6 column volume (CV) of ethanol, 4 CVof purified water, 2 CV of 5% HCl aq., 4 CV of purified water, 2 CV of2% NaOH aq. and 4 CV of purified water at the flow rate of 3 CV/hour.The oxidized peptide solution was then loaded to the column at 2 CV/h.After loading, the column was washed with 2 CV purified water at 2 CV/h.The elution was conducted by applying 80% ethanol aq. to the column at 2CV/h. The fractions with UV absorbents at 215 nm, was collected andcombined (125 mL). The combined fractions were then evaporated in vacuumto 10% of the original volume and the suspension was precipitated with125 mL of cold MTBE (10 vol). The solid was collected by filtration anddried in vacuum to yield the crude SP-304 (0.282 g, 55.0% HPLC).

Example 4: Oxidative Cyclization and Purification of SP-304 by RP-HPLC

Crude peptide (4-4), prepared as described in Example 2 was dissolved in10% ACN aqueous to an approximate concentration of 1.25 g/L withcontinuous stirring via a mechanical stirrer. The pH of the peptidesolution was adjusted to 8.5-9.0 with 20% ammonia aqueous and theresulting solution was stirred vigorously open to the atmosphere.Hydrogen peroxide (3%, 0.25 equiv.) was added and stirring was continuedat room temperature for 60 minutes. The HPLC analysis showed completeconsumption of the linear peptide. Then, the solution was acidified topH 3-4 with 10% AcOH aqueous. The resulting solution was diluted to aconcentration of about 1 g/L with purified water. Iodine (1.3% in ACN)was added in with vigorous stirring over a period of 10 minutes untilthe yellow color of the iodine persisted. At about half-hourly intervalssamples were taken from the mixture and analyzed by RP-HPLC. Themonocyclized peptide peak decreased gradually and a new peak (dicyclizedpeptide) emerged. Oxidation was complete when no monocyclized peptidepeak left. The excess iodine was neutralized by a small amount ofascorbic acid. The resulting solution was loaded on a C₁₈ RP-HPLC columnpacked with Kromasil 100 Å, 10 μm silica gel. After the dicyclizedpeptide solution was loaded, a 3 column volume of a solution of 90%mobile phase A (1.0% TEA, 0.5% H₃PO₄ in H₂O, pH=7) and 10% mobile phaseB (acetonitrile) was loaded to wash out lines. Then, gradient wasoperated from 10% B to 30% B in 80 minutes. Fractions were collected atrecorded intervals when main peak began to elute. The purity of eachfraction was monitored by analytical RP-HPLC. Fractions of purity ≤95%(not meeting the Main Pool criteria) were pooled accordingly and forwardprocessed using the same buffer system and gradient elution parametersstated above. All fractions with purity ≥95% were pooled and stored at2-8° C. The purified peptide solution was diluted in a 1:1 ratio withpurified water and then loaded to the same RP-HPLC column. Thecounter-ion exchange was accomplished by washing the column with 2-3column volumes of 0.5M ammonium acetate aqueous, followed by gradientelution from 90% C (0.2% AcOH aqueous solution) and 10% mobile phase D(ACN) to 50% mobile phase C and 50% mobile phase D in 50 minutes.Fractions were collected at recorded intervals and monitored byanalytical RP-HPLC. The fractions (≥95%) were collected and lyophilizedto obtain final dry peptide, 68.0 g (96.1% pure).

Example 5: Desalination and Isolation of SP-304 after Purification byRP-HPLC

After plecanatide was purified by RP-HPLC as described in Example 4, itwas desalinated and isolated. Briefly, the purified plecanatide inammonium acetate/acetonitrile/water buffer was loaded onto a columnpacked with polymeric absorbent (macroporous adsorption resin) and theneluted by a mixture of alcohol/water. Finally, the peptide alcoholsolution was concentrated under reduced pressure, precipitated with anether, e.g., diethyl ether or MTBE, and dried under vacuum to give thefinal product.

Resin (Polymeric Adsorbents) Screening

Resin Pre-Treatment:

Polymeric adsorbents, DA201-C(from Jiangsu Suqing, China; crosslinkedpolystyrene; surface area 1200-1400 m²/g; average pore diameter: 3-4 nm;pore volume: 1.1-1.2 ml/g; bulk density: 0.68-0.75 g/ml; specificdensity: 1.03-1.1 g/ml; moisture: 50-60%; particle size: 0.315-1.25 mm≥95%; effective diameter: 0.4-0.7 mm; uniformity coefficient: <1.6%),DA201-H (from Jiangsu Suqing, China; crosslinked polystyrene; surfacearea ≥800 m²/g; average pore diameter: 6-8 nm; pore volume: 1.5-1.8ml/g; bulk density: 0.65-0.70 g/ml; specific density: 1.02-1.07 g/ml;moisture: 55-65%; particle size: 0.315-1.25 mm ≥95%; effective diameter:0.4-0.7 mm; uniformity coefficient: <1.6%), ADS-5 (from Nankai Hecheng,China; crosslinked polystyrene; surface area 520-600 m²/g; average porediameter: 25-30 nm; bulk density: 0.7-0.8 g/ml; moisture: 60-70%;particle size: 0.315-1.25 mm ≥95%; uniformity coefficient: <1.6%), andADS-8 (from Nankai Hecheng, China; crosslinked polystyrene; surface area450-500 m²/g; average pore diameter: 12-16 nm; bulk density: 0.65-0.75g/ml; moisture: 60-70%; particle size: 0.315-1.25 mm 295%; uniformitycoefficient: <1.6%) were suspended in 4-6 volume of ethanol overnight.Decant or suction off the supernatant from the settled resin. Add 6-8volume of deionized water and resuspend the resin with gentle overheadstirring. Again, decant or suction off the supernatant from the settledresin. Repeat the above water treatment and decantation steps untilfines appearance is minimal.

Column Packing and Regeneration:

Resuspend the pre-treated resins above with 1-2 volume of deionizedwater to form the resin slurry respectively by using gentle agitation.Pour the resin slurry slowly down the inside of the column to preventair entrapment. After the resin slurry has been fully transferred to thecolumn, rinse the inside of the column using a squirt bottle containingdeionized water. Open the column outlet to from a settled resin bed(ID=4 cm, H=10 cm). Then the resin beds were washed successively at aflow rate of 3 CV per hour by 4 CV of deionized water, 2 CV of 5% HClaq, 4 CV of deionized water, 2 CV of 2% NaOH aq and finally 4 CV ofdeionized water till the pH of the elute was around 7.

Preparation of Loading Samples:

2000 mg of lyophilized plecanatide was dissolved in a mixture of 60 mLof ACN and 150 mL of 0.2% AcOH aq (the pH of the AcOH aq was adjusted to4 with 10% ammonia aq.). After filtration with 1.2 m Nylon membrane, thefiltrate was diluted to 250 mL with 0.2% AcOH aq (pH4) and split into 4parts (62.5 mL each) for loading.

Loading the Sample to the Columns:

62.5 mL of peptide solution above was loaded onto the above 4 columns ata flow rate of 2 CV/h respectively. The loading elute was collected andtested by RP-HPLC to evaluate the absorbent capacity of each resin. Theabsorbent capacity results of each resin were demonstrated in Table Xbelow.

TABLE X Peptide in Resins loading sample Peptide absorbed Absorbentratio DA201-C 500 mg 303.5 mg 60.7% DA201-H 500 mg 493.4 mg 98.7% ADS-5500 mg 450.5 mg 90.1% ADS-8 500 mg 466.1 mg 93.2%

HPLC Method:

HPLC machine: Shimadzu LC-10AD vp; column: Kromasil, C18, 4.6×250 mm;mobile phase A: 0.1% TFA in water; mobile phase B: 0.1% TFA in ACN;detect at: 215 nm; column temperature: 40° C.; flow rate: 1.0 mL/min;gradient: 25% B to 45% B in 30 min.

Absorbent Capacity Calculation:

The absorbent capacity of each resin was demonstrated by the absorbentratio of the peptide loaded onto each column, which was calculated bythe quantity of the peptide absorbed in each resin column divided by thepeptide quantity in each loading sample (500 mg). The quantity ofpeptide absorbed in each column was calculated by the formula below:

Quantity of peptide absorbed=Quantity of peptide in the loadingsample-Quantity of peptide in the loading elute=500 mg−62.5 mL×(1.6mg/mL×HPLC peak area of the eluate/HPLC peak area of the peptidestandard solution)

Washing the Column with Deionized Water:

The loaded columns above were then washed with 2 CV of deionized waterat 2 CV/h to remove the salts. The washing eluates were collected andanalyzed by RP-HPLC to determine the peptide quantity desorbed by waterusing the same method above. The desorbed peptide ratios of each resinwere listed in Table XI below.

TABLE XI Desorption Resins Peptide absorbed in resin Peptide desorbedratio DA201-C 303.5 mg 184.5 mg  60.8% DA201-H 493.4 mg 40.9 mg 8.3%ADS-5 450.5 mg 41.9 mg 9.3% ADS-8 466.1 mg 40.1 mg 8.6%

Desorbing the Peptide with 90% Ethanol/Water:

After 2 CV of water washes, the peptide absorbed in each column was theneluted by 1-2 CV of 90% ethanol in water at 2 CV/h. The elution wascollected and analyzed by RP-HPLC to determine the peptide quantitydesorbed by 90% ethanol using the same method above. The desorbedpeptide ratios of each resin were listed in Table XII below.

TABLE XII Peptide absorbed in resin after 2CV water Desorption Resinswashes Peptide desorbed ratio DA201-C 119.0 mg  47.6 mg (by 2CV ethanol)40% DA201-H 452.5 mg 452.5 mg (by 1.5CV ethanol) 100% ADS-5 408.6 mg408.6 mg (by 1.5 CV ethanol) 100% ADS-8 426.0 mg 426.0 mg (by 1.5 CVethanol) 100%

Isolation of the Peptide from the Ethanol Solution:

The collected peptide/ethanol/water solution from each column wasconcentrated under reduced pressure, precipitated with MTBE, filteredand dried in vacuo to give the final product. The overall yield of thepeptide processed by each column was demonstrated in Table XIII below.

TABLE XIII Overall Resins Peptide in loading sample Final productobtained yield DA201-C 500 mg  52 mg 10.4% DA201-H 500 mg 460 mg 92.0%ADS-5 500 mg 400 mg 80.0% ADS-8 500 mg 430 mg 86.0%

Resin Screening Conclusion:

From the data above (Table X to Table XIII), the DA201-H resin presentedthe best absorbent capacity and the best desorption (by ethanol)performance for plecanatide among the resins in the experiment.

Desalination and Isolation Process Optimization

Eluting Solvents Selection:

Isopropanol and ethanol are two commonly used solvents for eluting thepeptide from the polymeric absorbents. Table XIV shows the amount ofplecanatide that is able to be dissolved in aqueous ethanol orisopropanol solution depending on the v/v % isopropanol (or ethanol):water.

TABLE XIV Solvent Solubility 90% IPA/Water  67.5 mg/mL 75% IPA/Water596.1 mg/mL 50% IPA/Water 635.0 mg/mL 90% EtOH/Water 302.0 mg/mL 75%EtOH/Water 700.0 mg/mL

The water in the peptide/alcohol solution can be removed by azeotropicdistillation. Table XV shows the property of the binary azeotropes ofethanol/water and isopropanol/water.

TABLE XV Boiling Boiling Azeotrope Azeotrope Component A Component BPoint A Point B Boiling Point Wt. % A Water Ethanol 100° C. 78.3° C.78.2° C.   4% Water Isopropanol 100° C. 82.3° C. 80.3° C. 12.6%

Degradation of plecanatide will occur during the long time storage ofthe peptide/alcohol/water solution and the concentration process. TableXVI demonstrates the stability data of plecanatide in 75% IPA/watersolution and 90% EtOH/water at 23° C.

TABLE XVI Duration Purity (in 90% EtOH aq.)* Purity (in 75% IPA aq.)* 0hours 98.5% 98.7% 2 hours 98.4% 98.7% 4 hours N/A 98.3% 6 hours 98.1%97.5% 8 hours N/A 96.6% 10 hours  95.5% N/A 24 hours  92.0% N/A 25hours  N/A 96.1% *HPLC Method: HPLC machine: Shimadzu LC-10AD vp:column: Kromasil, C18, 4.6 × 250 mm; mobile phase A: 0.1% TFA in water;mobile phase B: 0.1% TFA in ACN; detect at: 215 nm; column temperature:40° C.; flow rate: 1.0 mL/min; gradient: 25% B to 45% B in 30 min.

From the testing data obtained, plecanatide was fairly stable at 23° C.in alcohol/water solution within 6 hours.

Elution experiments were conducted using isopropanol/water mixtures withdifferent v/v %. The desorption ratio and water content of the peptideeluates were listed in Table XVII below.

TABLE XVII v/v % IPA/Water 75% IPA/water 95% IPA/water 100% IPA/waterWater content in 65.9% 53.0% 49.8% peptide Desorption ratio  100%  100%  90%

75% Isopropanol/Water as Eluting Solution:

500 mg of plecanatide (98.1% pure) was dissolved in a mixture of 16 mLACN and 49 mL of 0.2% AcOH aqueous (the pH of the 0.2% AcOH solution wasadjusted to 4.0 by addition of 10% NH₄OH aq). After filtration by 1.2 mnylon membrane, the peptide solution was loaded onto a column packedwith DA201-H resin (ID=4 cm, H=10 cm, packed and pre-treated by theprocedure mentioned previously) at 2 CV/h. After loading, the column waswashed with 2 CV deionized water at 2 CV/h. Then, the peptide was elutedby 1.5 CV of 75% IPA/water at 2 CV/h. The elution was monitored byRP-HPLC. The eluate was collected (124 mL, 98.3% pure). Karl Fisheranalysis indicated water (65.9% wt. %). Into a 1-neck, 500-mL roundbottom flask was placed 124 mL of peptide/IPA/water eluate collectedabove (97.6% pure, slight degradation occurred during storage at 2-8° C.for 2 days). The flask was then placed under reduced pressure (60 Pa) ona rotary evaporator and partially immersed in a 23° C. water bath. Awhitish suspension was formed by feed-stripping approximately 622 mL ofisopropanol in 2 hours to about ⅓ of the initial volume of the solution(97.7% pure). Karl Fisher analysis indicated water (0.17% wt. %). To theconcentrate was added 350 mL of pre-chilled diethyl ether, the solid wascollected by centrifugation at 3500 rpm for 3 minutes and dried undervacuum to yield 333 mg of final product (yield 66.6%, 97.2% HPLCpurity).

95% Isopropanol/Water as Eluting Solution I:

500 mg of plecanatide (98.1% pure) was dissolved in a mixture of 16 mLACN and 49 mL of 0.2% AcOH aqueous (the pH of the 0.2% AcOH solution wasadjusted to 4.0 by addition of 10% NH₄OH aq). After filtration by 1.2 mnylon membrane, the peptide solution was loaded onto a column packedwith DA201-H resin (ID=4 cm, H=10 cm, pre-treated by the procedurementioned previously) at 2 CV/h. After loading, the column was washedwith 2 CV deionized water at 2 CV/h. Then, the peptide was eluted by 1.5CV of 95% IPA/water at 2 CV/h. The elution was monitored by RP-HPLC. Theeluate was collected (117 mL, 98.1% pure). Karl Fisher analysisindicated water (52% wt. %). Into a 1-neck, 500-mL round bottom flaskwas placed 117 mL of peptide/IPA/water eluate collected above. The flaskwas then placed under reduced pressure (50 Pa) on a rotary evaporatorand partially immersed in a 23° C. water bath. A whitish solid wasformed by feed-stripping approximately 470 mL of isopropanol in 130 min(97.9% pure). To the solid above was added 50 mL of pre-chilled diethylether to form a suspension and evaporated under reduced pressure (50 Pa)on a rotary evaporator at 23° C. to dryness. Yield was 430 mg of finalproduct (86%). HPLC purity was 97.9%.

95% Isopropanol/Water as Eluting Solution II:

500 mg of plecanatide (98.1% pure) was dissolved in a mixture of 16 mLACN and 49 mL of 0.2% AcOH aqueous (the pH of the 0.2% AcOH solution wasadjusted to 4.0 by addition of 10% NH₄OH aq). After filtration by 1.2 mnylon membrane, the peptide solution was loaded onto a column packedwith DA201-H resin (ID=4 cm, H=10 cm, packed and pre-treated by theprocedure mentioned previously) at 2 CV/h. After loading, the column waswashed with 2 CV deionized water at 2 CV/h. Then, the peptide was elutedby 1.5 CV of 95% IPA/water at 2 CV/h. The elution was monitored byRP-HPLC. The eluate was collected (118 mL, 98.2% pure). Karl Fisheranalysis indicated water (53% wt. %). Into a 1-neck, 500-mL round bottomflask was placed 118 mL of peptide/IPA/water eluate collected above. Theflask was then placed under reduced pressure (50 Pa) on a rotaryevaporator and partially immersed in a 23° C. water bath. A whitishsuspension (˜40 mL) was formed by feed-stripping approximately 330 mL ofisopropanol in 100 min (97.7% pure). To the suspension above was added400 mL of pre-chilled diethyl ether to form a suspension. After standingat ambient temperature for 1 hour, the solid was collected bycentrifugation at 3500 rpm for 3 minutes, and dried under vacuum toyield 370 mg of final product. Yield was 74%. HPLC purity was 97.9%.

95% Isopropanol/Water as Eluting Solution III:

10 g of plecanatide was desalted and precipitated in a manner similar tothat described above. Interestingly, the precipitation yield wasimproved to 93%, which is a significant increase in yield. HPLC purityafter precipitation was 98.47%.

Example 6: Characterization of Lyophilized SP-304 and PrecipitatedSP-304

The plecanatide purified by lyophilization as described in Example 4 andplecanatide purified by precipitation as described in Example 5 wereanalyzed to determine significant chemical impurity values such as thelevels of acetamide, TFA, ammonium ion, and acetonitrile. The resultsare listed in the table below.

Acetamide TFA Ammonium ion Acetonitrile Lyophilized 356 ppm 0.14% 1.58%40 ppm plecanatide Precipitated <28 ppm (LOQ 0.09% 0.23% Not Detectedplecanatide of method) (20 ppm LOQ)

As demonstrated by results above, the precipitation process providedsignificantly reduced levels of undesirable process impurities.

The plecanatide purified by lyophilization as described in Example 4 andplecanatide purified by precipitation as described in Example 5 weremeasured to obtain their bulk densities, tap densities, particle sizedistribution, and shape.

Equipment:

(1) Tap Density Tester Model TD-1020; (2) Sonic Sifter Separator ModelL3P; (3) Optical Microscope LINITRON 2850.

Methods:

1) Bulk and Tap Density Measurements: Modified USP 1 Method

a. 100.0 mL graduate cylinder was used for lyophilized plecanatide

b. 10.0 mL graduated cylinder was used for precipitated plecanatide

2) Particle Size Distribution Analysis

a. Screens used: 200, 140, 100, 60, 40 and 30 meshes.

b. Sample size: 2 g of lyophilized plecanatide and 6.4 g of precipitatedplecanatide

3) Optical Microscopic Analysis: Particle Size and Shape

a. Dry powder was manually dispersed onto a microscopic plate

b. Magnification: 100×

c. Under normal light condition (no polarized filters)

Results:

(1) Physical Appearance: lyophilized plecanatide is a light, fluffy andwhite powder. Precipitated plecanatide is a slightly off-white powder.

(2) Bulk and Tap Density: Table XVIII provides bulk and tap density datafor Plecanatide samples of both lyophilized and precipitated:

TABLE XVIII Plecanatide Sample Bulk Density, g/mL Tap Density, g/mLLyophilized, Lot 101221 0.0332 0.0680 Precipitated, Lot 120210 0.4860.641

As seen from the data, the precipitated plecanatide is unexpectedlysignificantly denser than the lyophilized plecanatide. The precipitatedplecanatide has less tendency of dust generation during processing,which affords the advantages of increased safety and reduced processinglosses.

(3) Particle Size Distribution: Table XIX summarizes the particle sizedistribution analysis. FIG. 1 presents the data graphically.

TABLE XIX # Particle Mesh Size Weight Retained (g) Percent Retained Size(μm) Lyophilized Precipitated Lyophilized Precipitated 30 600 0.07 0.453.6% 7.1% 40 425 0.26 0.17 13.3% 2.7% 60 250 0.17 0.67 8.7% 10.5% 100150 0.51 2.08 26.0% 32.7% 140 106 0.65 1.22 33.2% 19.2% 200 75 0.24 0.6812.2% 10.7% Pan <75 0.06 1.09 3.1% 17.1% Total 1.96 6.36 100.0% 100.0%

As demonstrated by Table XIX and FIG. 1, the particle size distributionsare different for both types of plecanatide. During analysis, it wasobserved that the precipitated plecanatide contained some largerparticles, which could be broken up easily. It was also noticed that thelyophilized plecanatide appeared to be flaky and sticking onto top andbottom of sieves, whereas no sticking was observed for the precipitatedplecanatide. It indicates a better processing property of theprecipitated plecanatide.

(4) Particle Size and Shape: FIGS. 2 and 3 provide optical microscopicanalysis of samples of lyophilized and precipitated plecanatide,respectively. As seen in FIG. 2, the lyophilized plecanatide is inamorphous form and has irregular shapes of particles. They form physicalaggregates with particles lying on top of each other. In FIG. 3, theprecipitated plecanatide shows distinguishable individual particles.From the particle appearances and shapes, the precipitated plecanatidewill have better flow property and therefore can facilitate solidprocessing during manufacturing.

The lyophilized and precipitated forms of plecanatide have showndistinguishable physical appearances and properties by density, particlesize distribution and shape analyses. The precipitated form is moresuitable for solid dosage form processing during manufacturing in termsof reducing dust generation, less sticking onto processing equipment,and potentially less processing losses.

The precipitated form is more suitable for processing solid dosage formduring manufacture (e.g., a low-dose solid dosage form). The higherdensity of the precipitated material will reduce aerosol or “dust”losses during weighing, transferring, and blending. The differentparticle shape has been shown to reduce loss caused by sticking toscreens or sieves. The higher density should improve content uniformitysince the size and density of the drug particles more closely matchthose of the excipients.

Example 7: Low-Dose Formulation of Precipitated SP-304

The plecanatide purified by precipitation as described in Example 5 isprocessed further to make low-dose formulations as described below.

composition of dry-blending batch Concentration % Item No. Ingredientw/w 1 SP-304 0.3246 2 Microcrystalline 99.43 cellulose (Avicel PH 102) 3Magnesium stearate 0.2500 4 HPMC capsule shells n/a Total 100

Blending:

Avicel PH 102 is screened through a 60 mesh screen. V-blenders (1 Qt, 4Qt, and 16 Qt) are then dusted by the screened Avicel PH 102. SP-304 isscreened through a 200 mesh screen and loaded into the 1-Qt V-blender.Then, about 80 g Avicel PH 102 is added into the 1-Qt blender and themixture is blended for 10 minutes at 25 rpm. The mixture is thentransferred to the 4-Qt V-blender which is pre-dusted by the screenedAvicel PH 102. The 1-Qt blender is rinsed with Avicel and the rinsematerial is transferred to the 4-Qt blender. The rinsing is repeateduntil all SP-304 is transferred to the 4-Qt blender. About 200 g Avicelis added to the 4-Qt V-blender and the mixture is blended for 10minutes. The resulting blend is then screened through a 60 mesh screenand then transferred into the pre-dusted 16-Qt blender (dusted with 1500g Avicel). The 4-Qt blender is rinsed with Avicel and the rinse materialis transferred to the 16-Qt blender. The remaining Avicel is added tothe 16-Qt blender and the mixture is blended for 10 minutes. Theresulting blend is passed through Comil, rinsed with excess of Avicel,and then returned to the 16-Qt blender and is further blended for 5minutes. Proper amount of magnesium stearate is weighed, screenedthrough a 60 mesh screen, and added into the 16-Qt blender. Theresulting mixture is blended for 2 minutes. The resulting mixture isthen either packaged in capsules or compressed to form tablets.

Encapsulation

A MG2 Planeta capsule filler is set up. Average weight of the emptycapsule shells is determined and target capsule fill weight wascalculated (±5%). The blend from the above process is added into thehopper of the capsule filler and encapsulation is started. Run weightparameters are manually adjusted. Resulting capsules are then sortedaccording to the target fill weight.

Compression

A Fette tablet press is set up. Then the blend mixture is loaded intothe powder hopper and tooling is installed. The weight of each tablet isset to be 100 mg±5% and hardness to be 4-6 Kp. The weight, hardness, andthickness of tablets are measured and recorded every 5 to 10 minutes.Friability measurement is also performed to ensure satisfactory product.

We claim:
 1. A process of preparing a peptide comprising a GCC agonistsequence selected from the group consisting of SEQ ID NOs: 1-249,wherein the GCC agonist sequence is n amino acid units in length, withthe N-terminal unit at position 1 and the C-terminal unit at position n,the process comprising: providing a first fragment having a firstsequence of amino acid units from position j through position k of theGCC agonist sequence, wherein j is an integer between 1 and n−1, k is aninteger between 2 and n and is greater than j, and the first fragment isprotected except for an amino group of the amino acid unit at positionj, or alternatively, a carboxyl group of the amino acid unit at positionk, providing a second fragment having a second sequence of amino acidunits from position h through position j−1 of the GCC agonist sequenceor having a third sequence of amino acid units from position k+1 throughposition m of the GCC agonist sequence, wherein h is an integer between1 and n−2 and is smaller than j, m is an integer between k+2 and n, andthe second fragment is protected except for a carboxyl group of theamino acid unit at position j−1 or an amino group of the amino acid unitat position k+1, and coupling the first and the second fragments via asolution-phase synthesis to yield a protected peptide having a sequenceof amino acid units from position h through position k of the GCCagonist sequence or a protected peptide having a sequence of amino acidunits from position j through position m of the GCC agonist sequence. 2.The process of claim 1, wherein the GCC agonist sequence is selectedfrom the group consisting of SEQ ID NOs: 1, 8, 9, 55, 56, 58, and
 59. 3.The process of claim 1, wherein the GCC agonist sequence is selectedfrom the group consisting of SEQ ID NOs: 1 and
 9. 4. The process ofclaim 3, wherein at least one of the first and second fragments isprovided via a solid-phase peptide synthesis.
 5. The process of claim 4,wherein the solid-phase peptide synthesis is a Fmoc solid-phasesynthesis.
 6. The process of claim 5, wherein the Fmoc solid-phasesynthesis is performed on 2-chlorotrityl resin.
 7. The process of claim3, wherein h is 1, j is 7, and k is
 16. 8. The process of claim 3,wherein h is 7, j is 15, and k is
 16. 9. The process of claim 8, furthercomprising deprotecting an amino group of the amino acid unit atposition 7 of the protected peptide having the sequence of amino acidunits from position 7 through position 16 of the GCC agonist sequence toyield a position-7 reactive peptide.
 10. The process of claim 9, furthercomprising providing a third fragment having a sequence of amino acidunits from position 1 through position 6 of the GCC agonist sequence,wherein the third fragment is protected except for a carboxyl group ofthe amino acid at position
 6. 11. The process of claim 10, wherein thethird fragment is provided via a solid-phase peptide synthesis.
 12. Theprocess of claim 11, wherein the solid-phase peptide synthesis is Fmocsolid-phase synthesis.
 13. The process of claim 10, further comprisingcoupling the third fragment and the position-7 reactive peptide via asolution-phase synthesis to yield a protected linear peptide having asequence of amino acid units from position 1 through position 16 of theGCC agonist sequence.
 14. The process of claim 13, further comprisingdeprotecting the protected linear peptide to yield a deprotected linearpeptide.
 15. The process of claim 14, further comprising oxidizing thedeprotected linear peptide to yield the peptide comprising the GCCagonist sequence selected from the group consisting of SEQ ID NOs: 1 and9.
 16. The process of claim 1, wherein at least one of the first andsecond fragments is provided via a solid-phase peptide synthesis. 17.The process of claim 16, wherein the solid-phase peptide synthesis isFmoc solid-phase synthesis.
 18. The process of claim 1, wherein each ofthe first and second fragments is not more than 10 amino acid units inlength.
 19. The process of claim 18, wherein the second fragment has thesecond sequence of amino acid units from position h through position j−1of the GCC agonist sequence and either one or both of the amino acidunit of the second fragment at position j−1 and the amino acid unit ofthe first fragment at position k is selected from the group consistingof glycine, proline, leucine, alanine, and arginine.
 20. The process ofclaim 18, wherein the second fragment has the second sequence of aminoacid units from position h through position j−1 of the GCC agonistsequence and either one or both of the amino acid unit of the secondfragment at position j−1 and the amino acid unit of the first fragmentat position k is selected from the group consisting of glycine andproline.
 21. The process of claim 18, wherein the second fragment hasthe third sequence of amino acid units from position k+1 throughposition m of the GCC agonist sequence and either one or both of theamino acid unit of the second fragment at position m and the amino acidunit of the first fragment at position k is selected from the groupconsisting of glycine, proline, leucine, alanine, and arginine.
 22. Theprocess of claim 18, wherein the second fragment has the third sequenceof amino acid units from position k+1 through position m of the GCCagonist sequence and either one or both of the amino acid unit of thesecond fragment at position m and the amino acid unit of the firstfragment at position k is selected from the group consisting of glycineand proline.
 23. The process of claim 1, wherein k is n, the secondfragment has the second sequence of amino acid units from position hthrough position j−1 of the GCC agonist sequence, and the amino acidunit at positions j−1 is selected from the group consisting of glycine,proline, leucine, alanine, and arginine.
 24. The process of claim 23,further comprising deprotecting an amino group of the amino acid unit atposition h of the protected peptide having a sequence of amino acidunits from position h through position k of the GCC agonist sequence toyield a position-h reactive peptide.
 25. The process of claim 24,further comprising providing a fourth fragment having a sequence ofamino acid units from position 1 through position h−1 of the GCC agonistsequence, wherein the fourth fragment is protected except for a carboxylgroup of the amino acid unit at position h−1.
 26. The process of claim25, wherein the fourth fragment is provided via a solid-phase peptidesynthesis.
 27. The process of claim 26, wherein the solid-phase peptidesynthesis is Fmoc solid-phase synthesis.
 28. The process of claim 25,wherein the fourth fragment is not more than 10 amino acid units inlength.
 29. The process of claim 25, further comprising coupling thefourth fragment and the position-h reactive peptide via a solution-phasesynthesis to yield a protected linear peptide having a sequence of aminoacid units from position 1 through position n of the GCC agonistsequence.
 30. The process of claim 29, further comprising deprotectingthe protected linear peptide to yield a deprotected linear peptidehaving a sequence of amino acid units from position 1 through position nof the GCC agonist sequence.
 31. The process of claim 30, furthercomprising oxidizing the deprotected linear peptide to yield the peptidecomprising the GCC agonist sequence selected from the group consistingof SEQ ID NOs: 1-249.
 32. The process of claim 31, further comprisingpurifying the peptide comprising the GCC agonist sequence selected fromthe group consisting of SEQ ID NOs: 1-249, wherein the purificationcomprises adsorbing the peptide onto a polymeric adsorbent column,optionally rinsing the peptide with deionized water, eluting theoptionally rinsed peptide off the polymeric adsorbent column with analcohol aqueous solution to form a peptide solution, removing water andthe alcohol from the peptide solution to precipitate the peptide, andoptionally adding an ether to the dewatered peptide to facilitateprecipitation of the peptide.
 33. The process of claim 32, wherein thealcohol aqueous solution comprises isopropanol.
 34. The process of claim32, wherein the ether comprises diethyl ether.
 35. The process of claim32, further comprising salt exchanging the peptide by washing thepeptide with an aqueous solution comprising an ammonium salt before thepurification.
 36. The process of claim 35, further comprising,lyophilizing the peptide after the salt exchanging step before thepurification.
 37. A process of purifying the peptide comprising the GCCagonist sequence selected from the group consisting of SEQ ID NOs:1-249, the process comprising adsorbing the peptide onto a polymericadsorbent column, desalting the peptide, eluting the desalted peptideoff the polymeric adsorbent column with an alcohol aqueous solution toform a peptide solution, removing water from the peptide solution, andadding an ether to the dewatered peptide to precipitate the peptide. 38.The process of claim 37, wherein the alcohol aqueous solution comprisesisopropanol.
 39. The process of claim 37, wherein the ether comprisesdiethyl ether.
 40. The process of claim 37, further comprising saltexchanging the peptide by washing the peptide with an aqueous solutioncomprising an ammonium salt before the purification.
 41. The process ofclaim 40, further comprising, lyophilizing the peptide after the saltexchanging step before the purification.
 42. A purified peptidecomprising the GCC agonist sequence selected from the group consistingof SEQ ID NOs: 1-249, wherein the peptide has a bulk density of no lessthan 0.1 g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no lessthan 0.4 g/mL, or no less than 0.5 g/mL.
 43. A purified peptide, whereinthe peptide is purified by the process of any of claims 37-41.
 44. Thepurified peptide of claim 42, wherein the peptide is purified by theprocess of any of claims 37-41.
 45. The purified peptide of any ofclaims 42-44, wherein the peptide has a tap density of no less than 0.1g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4g/mL, no less than 0.5 g/mL, or no less than 0.6 g/mL.
 46. The purifiedpeptide of any of claims 42-45, wherein the peptide has achromatographic purity of no less than 95%, no less than 97%, or no lessthan 98%.
 47. The purified peptide of any of claims 42-46, whereinpeptide is substantially free of water.
 48. The purified peptide of anyof claims 42-47, wherein peptide is substantially free of impuritiesselected from acetonitrile, acetamide, alcohols, ammonium, and TFA. 49.The purified peptide of any of claims 42-48, wherein the peptide issubstantially free of topoisomers.
 50. The purified peptide of any ofclaims 42-49, wherein the peptide comprises the GCC agonist sequence ofSEQ ID NO:
 1. 51. The purified peptide of any of claims 42-49, whereinthe peptide comprises the GCC agonist sequence of SEQ ID NO: 9.